Non-Aggregating Fluorescent Proteins and Methods for Using the Same

ABSTRACT

Nucleic acid compositions encoding non-aggregating chromo/fluoroproteins and mutants thereof, as well as the proteins encoded by the same, are provided. The proteins of interest are polypeptides that are non-aggregating colored and/or fluorescent proteins, where the non-aggregating feature arises from the modulation of residues in the N-terminus of the protein and the chromo and/or fluorescent feature arises from the interaction of two or more residues of the protein. Also provided are fragments of the subject nucleic acids and the peptides encoded thereby, as well as antibodies to the subject proteins and transgenic cells and organisms. The subject protein and nucleic acid compositions find use in a variety of different applications. Finally, kits for use in such applications, e.g., that include the subject nucleic acid compositions, are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part of application Ser. No.10/006,922 filed on Dec. 4, 2001 and also claims priority to applicationSer. No. 60/270,983 filed on Feb. 21, 2001; the disclosures of whichapplications are incorporated in their entirety herein.

INTRODUCTION

1. Field of the Invention

The field of this invention is chromoproteins and fluorescent proteins.

2. Background of the Invention

Labeling is a tool for marking a protein, cell, or organism of interestand plays a prominent role in many biochemistry, molecular biology andmedical diagnostic applications. A variety of different labels have beendeveloped, including radiolabels, chromolabels, fluorescent labels,chemiluminescent labels, etc. However, there is continued interest inthe development of new labels. Of particular interest is the developmentof new protein labels, including chromo- and/or fluorescent proteinlabels.

An important new class of fluorescent proteins that have recently beendeveloped are the Reef Coral Fluorescent Proteins, as described in Matz,M. V., et al. (1999) Nature Biotechnol., 17:969-973. While thesefluorescent proteins exhibit many positive attributes, certain versionsare prone to high molecular weight aggregation, which can pose problemsand consequently limit their applicability.

As such, there is intense interest in the development of non-aggregatingversions of this important new class of fluorescent proteins. Thepresent invention satisfies this need.

3. Relevant Literature

U.S. patents of interest include: U.S. Pat. Nos. 6,066,476; 6,020,192;5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;5,874,304; and 5,491,084. International Patent Publications of interestinclude: WO 00/46233; WO 99/49019; and DE 197 18 640 A. Also of interestare: Anderluh et al., Biochemical and Biophysical ResearchCommunications (1996) 220:437-442; Dove et al., Biological Bulletin(1995) 189:288-297; Fradkov et al., FEBS Lett. (2000) 479(3):127-30;Gurskaya et al., FEBS Lett., (2001) 507(1):16-20; Gurskaya et al., BMCBiochem. (2001) 2:6; Lukyanov, K., et al (2000) J Biol Chemistry275(34):25879-25882; Macek et al., Eur. J. Biochem. (1995) 234:329-335;Martynov et al., J Biol Chem. (2001) 276:21012-6; Matz, M. V., et al.(1999) Nature Biotechnol., 17:969-973; Terskikh et al., Science (2000)290:1585-8; Tsien, Annual Rev. of Biochemistry (1998) 67:509-544; Tsien,Nat. Biotech. (1999) 17:956-957; Ward et al., J. Biol. Chem. (1979)254:781-788; Wiedermann et al., Jarhrestagung der Deutschen Gesellschactfur Tropenokologie-gto. Ulm. 17-19. February 1999. Poster P-4.20;Yanushevich et al., FEBS Lett (Jan. 30, 2002) 511(1-3):11-4, andYarbrough et al., Proc Natl Acad Sci USA (2001) 98:462-7.

SUMMARY OF THE INVENTION

Nucleic acid compositions encoding non-aggregating chromo/fluoroproteinsand mutants thereof, as well as the proteins encoded by the same, areprovided. The proteins of interest are polypeptides that arenon-aggregating colored and/or fluorescent proteins, where thenon-aggregating feature arises from the modulation of residues in theN-terminus of the protein and the chromo and/or fluorescent featurearises from the interaction of two or more residues of the protein. Alsoprovided are fragments of the subject nucleic acids and the peptidesencoded thereby, as well as antibodies to the subject proteins andtransgenic cells and organisms. The subject protein and nucleic acidcompositions find use in a variety of different applications. Finally,kits for use in such applications, e.g., that include the subjectnucleic acid compositions, are provided.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 provides the nucleotide and amino acid sequence of wild typeamFP486 (NFP-1). (SEQ ID NO:01 & 02)

FIG. 2 provides the nucleotide and amino acid sequence of wild typezFP506 (NFP-3). (SEQ ID NO:03 & 04)

FIG. 3 provides the nucleotide and amino acid sequence of wild typezFP538 (NFP-4). (SEQ ID NO:05 & 06)

FIG. 4 provides the nucleotide and amino acid sequence of wild typedrFP583 (NFP-6). (SEQ ID NO: 07 & 08); as well as the nucleotide andamino acid sequence of an alternative version thereof.

FIG. 5 provides the nucleotide and amino acid sequence of wild typeasFP600 (NFP-7). (SEQ ID NO:09 & 10)

FIG. 6 provides the nucleotide and amino acid sequence of 6/9Q hybridprotein. (SEQ ID NO:11 & 12)

FIG. 7 provides the nucleotide sequence of mutant E57-NA (DsRED2) (SEQID NO:13)

FIG. 8 provides the nucleotide sequence of mutant E5-NA (Timer NA). (SEQID. NO:14)

FIG. 9 provides the nucleotide and amino acid sequence of FP3-NA (SEQ IDNO:15 & 16)

FIG. 10 provides the nucleotide and amino acid sequence of NFP4-NA (SEQID NO:17 & 18).

FIG. 11 provides the nucleotide and amino acid sequence of mut32-NA (SEQID NO: 19 &20)

FIG. 12 provides the nucleotide and amino acid sequence of mutant FP7-NA(SEQ ID NO:21 & 22).

FIG. 13 provides the nucleotide and amino acid sequence of mutant FP7-NAdimer. (SEQ ID NO:23 & 24).

FIG. 14. Multiple alignment of N-terminal regions of the fluorescentproteins. The arrow above the sequences represents the first β-sheet inthe proteins, based on GFP and drFP583 structures. Amino acid residuessubstituted in the non-aggregating mutants are shaded.

FIG. 15. Pseudo-native gel-electrophoresis of parental fluorescentproteins (odd lanes) and their non-aggregating mutants (even lanes). Thephotograph was taken under UV illumination. Aggregated proteins areobserved in the stacking gel. Oligomeric proteins migrate through theseparating gel as bands of high molecular weights (expected MW of FPmonomers and tetramers are about 27 and 108 kDa). Molecular weightstandards are shown on the left of the gel. Lanes: 1—DsRed mutant E57;2—E57-NA; 3—DsRed mutant Timer; 4—Timer-NA; 5—ds/drFP616;6—ds/drFP616-NA; 7—zFP506 mutant N66M; 8—zFP506-N66M-NA; 9—zFP538 mutantM129V; 10—zFP538-M129V-NA; 11—amFP486 mutant K68M; 12—amFP486-K68M-NA;13—asFP595 mutant M35-5; 14—M35-5-NA; 15—EGFP.

FIG. 16. Fluorescence images of cells expressing parental (left column)and corresponding non-aggregating (right column) fluorescent proteins(see Table 2 for details). Protein names are shown on the left.EGFP-expressing cells are shown for comparison (below) as a well-knownnon-aggregating fluorescent protein.

DEFINITIONS

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and it (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” (B. D. Hames & S. J. Higgins eds. (1985)); “Transcriptionand Translation” (B. D. Hames & S. J. Higgins eds. (1984)); “Animal CellCulture” (R. I. Freshney, ed. (1986)); “Immobilized Cells and Enzymes”(IRL Press, (1986)); B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded formor a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes.

A DNA “coding sequence” is a DNA sequence which is transcribed andtranslated into a polypeptide in vivo when placed under the control ofappropriate regulatory sequences. The boundaries of the coding sequenceare determined by a start codon at the 5′ (amino) terminus and atranslation stop codon at the 3′ (carboxyl) terminus. A coding sequencecan include, but is not limited to, prokaryotic sequences, cDNA fromeukaryotic mRNA, genomic DNA sequences from eukaryotic (e.g., mammalian)DNA, and synthetic DNA sequences. A polyadenylation signal andtranscription termination sequence may be located 3′ to the codingsequence.

As used herein, the term “hybridization” refers to the process ofassociation of two nucleic acid strands to form an antiparallel duplexstabilized by means of hydrogen bonding between residues of the oppositenucleic acid strands.

The term “oligonucleotide” refers to a short (under 100 bases in length)nucleic acid molecule.

“DNA regulatory sequences”, as used herein, are transcriptional andtranslational control sequences, such as promoters, enhancers,polyadenylation signals, terminators, and the like, that provide forand/or regulate expression of a coding sequence in a host cell.

A “promoter sequence” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. For purposes of defining the presentinvention, the promoter sequence is bounded at its 3′ terminus by thetranscription initiation site and extends upstream (5′ direction) toinclude the minimum number of bases or elements necessary to initiatetranscription at levels detectable above background. Within the promotersequence will be found a transcription initiation site, as well asprotein binding domains responsible for the binding of RNA polymerase.Eukaryotic promoters will often, but not always, contain “TATA” boxesand “CAT” boxes. Various promoters, including inducible promoters, maybe used to drive the various vectors of the present invention.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

A cell has been “transformed” or “transfected” by exogenous orheterologous DNA when such DNA has been introduced inside the cell. Thetransforming DNA may or may not be integrated (covalently linked) intothe genome of the cell. In prokaryotes, yeast, and mammalian cells forexample, the transforming DNA may be maintained on an episomal elementsuch as a plasmid. With respect to eukaryotic cells, a stablytransformed cell is one in which the transforming DNA has becomeintegrated into a chromosome so that it is inherited by daughter cellsthrough chromosome replication. This stability is demonstrated by theability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the transformingDNA. A “clone” is a population of cells derived from a single cell orcommon ancestor by mitosis. A “cell line” is a clone of a primary cellthat is capable of stable growth in vitro for many generations.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. In another example, heterologous DNA includes coding sequencein a construct where portions of genes from two different sources havebeen brought together so as to produce a fusion protein product. Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

As used herein, the term “reporter gene” refers to a coding sequenceattached to heterologous promoter or enhancer elements and whose productmay be assayed easily and quantifiably when the construct is introducedinto tissues or cells.

The amino acids described herein are preferred to be in the “L” isomericform. The amino acid sequences are given in one-letter code (A: alanine;C: cysteine; D: aspartic acid; E: glutamic acid; F: phenylalanine; G:glycine; H: histidine; I: isoleucine; K: lysine; L: leucine; M:methionine; N: asparagine; P: proline; Q: glutamine; R: arginine; S:serine; T: threonine; V: valine; W: tryptophan; Y: tyrosine; X: anyresidue). NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide. In keeping with standardpolypeptide nomenclature, J Biol. Chem., 243 (1969), 3552-59 is used.

The term “immunologically active” defines the capability of the natural,recombinant or synthetic chromo/fluorescent protein, or any oligopeptidethereof, to induce a specific immune response in appropriate animals orcells and to bind with specific antibodies. As used herein, “antigenicamino acid sequence” means an amino acid sequence that, either alone orin association with a carrier molecule, can elicit an antibody responsein a mammal. The term “specific binding,” in the context of antibodybinding to an antigen, is a term well understood in the art and refersto binding of an antibody to the antigen to which the antibody wasraised, but not other, unrelated antigens.

As used herein the term “isolated” is meant to describe apolynucleotide, a polypeptide, an antibody, or a host cell that is in anenvironment different from that in which the polynucleotide, thepolypeptide, the antibody, or the host cell naturally occurs.

Bioluminescence (BL) is defined as emission of light by living organismsthat is well visible in the dark and affects visual behavior of animals(See e.g., Harvey, E. N. (1952). Bioluminescence. New York: AcademicPress; Hastings, J. W. (1995). Bioluminescence. In: Cell Physiology (ed.by N. Speralakis). pp. 651-681. New York: Academic Press; Wilson, T. andHastings, J. W. (1998). Bioluminescence. Annu Rev Cell Dev Biol 14,197-230). Bioluminescence does not include so-called ultra-weak lightemission, which can be detected in virtually all living structures usingsensitive luminometric equipment (Murphy, M. E. and Sies, H. (1990).Visible-range low-level chemiluminescence in biological systems. Meth.Enzymol. 186, 595-610; Radotic, K, Radenovic, C, Jeremic, M. (1998.)Spontaneous ultra-weak bioluminescence in plants: origin, mechanisms andproperties. Gen Physiol Biophys 17, 289-308), and from weak lightemission which most probably does not play any ecological role, such asthe glowing of bamboo growth cone (Totsune, H., Nakano, M., Inaba, H.(1993). Chemiluminescence from bamboo shoot cut. Biochem. Biophys. ResComm. 194, 1025-1029) or emission of light during fertilization ofanimal eggs (Klebanoff, S. J., Froeder, C. A., Eddy, E. M., Shapiro, B.M. (1979). Metabolic similarities between fertilization andphagocytosis. Conservation of peroxidatic mechanism. J. Exp. Med. 149,938-953; Schomer, B. and Epel, D. (1998). Redox changes duringfertilization and maturation of marine invertebrate eggs. Dev Biol 203,1-11).

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Nucleic acid compositions encoding non-aggregating chromo/fluoroproteinsand mutants thereof, as well as the proteins encoding the same, areprovided. The proteins of interest are non-aggregating proteins that arecolored and/or fluorescent, where the non-aggregating feature arisesfrom the modulation of residues in the N-terminus of the proteins andthe chromo and/or fluorescent feature arises from the interaction of twoor more residues of the protein. Also of interest are proteins that aresubstantially similar to, or mutants of, the above specific proteins.Also provided are fragments of the nucleic acids and the peptidesencoded thereby, as well as antibodies to the subject proteins, andtransgenic cells and organisms that include the subject nucleicacid/protein compositions. The subject protein and nucleic acidcompositions find use in a variety of different applications. Finally,kits for use in such applications, e.g., that include the subjectnucleic acid compositions, are provided.

Before the subject invention is described further, it is to beunderstood that the invention is not limited to the particularembodiments of the invention described below, as variations of theparticular embodiments may be made and still fall within the scope ofthe appended claims. It is also to be understood that the terminologyemployed is for the purpose of describing particular embodiments, and isnot intended to be limiting. Instead, the scope of the present inventionwill be established by the appended claims.

In this specification and the appended claims, the singular forms “a,”“an” and “the” include plural reference unless the context clearlydictates otherwise. Unless defined otherwise, all technical andscientific terms used herein have the same meaning as commonlyunderstood to one of ordinary skill in the art to which this inventionbelongs.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range, and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood to one of ordinary skill inthe art to which this invention belongs. Although any methods, devicesand materials similar or equivalent to those described herein can beused in the practice or testing of the invention, the preferred methods,devices and materials are now described.

All publications mentioned herein are incorporated herein by referencefor the purpose of describing and disclosing the cell lines, vectors,methodologies and other invention components that are described in thepublications which might be used in connection with the presentlydescribed invention.

In further describing the subject invention, the subject nucleic acidcompositions will be described first, followed by a discussion of thesubject protein compositions, antibody compositions and transgeniccells/organisms. Next a review of representative methods in which thesubject proteins find use is provided.

Nucleic Acid Compositions

As summarized above, the subject invention provides nucleic acidcompositions encoding non-aggregating chromo- and fluoroproteins andmutants thereof, as well as fragments and homologues of these proteins.

As summarized above, the proteins encoded by the subject nucleic acidsare non-aggregating chromo and/or fluorescent proteins. Bynon-aggregating is meant that the proteins do not aggregate, i.e.complex with each other form high molecular weight aggregates. As usedherein, an “aggregate” refers to a higher order molecular complex, e.g.,a complex that comprises two or more tetramers of the protein. Themolecular weight of such aggregates typically exceeds about 100 kDa, andmore typically about 150 kDa. Aggregates are distinguished frommulitimers, where the term “multimer” refers to oligomers, such asdimers, trimers, and tetramers. Non-aggregating polypeptides of thesubject invention include polypeptides that show reduced aggregation invitro and/or in vivo as compared to their corresponding aggegratinganalogues, e.g., corresponding wild type proteins.

In certain embodiments, the subject polypeptides show decreasedaggregation in vitro relative to their corresponding aggregatinganalogues, e.g., their corresponding wild type proteins. “Reducedaggregation in vitro” refers to reduced aggregation in a cell-freesystem or in solution. In some embodiments, the non-aggregatingpolypeptide shows less than about 90%, less than about 80%, less thanabout 70%, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, or less than about 5% of theaggregation shown by the corresponding aggregating analogue under thesame in vitro conditions, e.g., less than about 60%, less than about50%, less than about 40%, less than about 30%, less than about 20%, lessthan about 10%, or less than about 5%, of the subject polypeptidepresent in a sample is aggregated. In vitro conditions suitable forcomparing a subject polypeptide with its corresponding aggregatinganalogue are conditions that do not prevent aggregation of theaggregating analogue, e.g., standard physiological conditions. Any of awide variety of buffer systems used in the art to study physiologicalphenomena can be used for in vitro comparisons. Non-limiting examples ofsuch conditions include, but are not limited to, a salt concentration inthe range of from about 0.01 mM to about 0.1 mM; a temperature in therange of from about 19° C. to about 25° C.; and a pH in the range offrom about 6.5 to about 8.0. Buffers that are suitable for comparison ofaggregation include, but are not limited to, any physiological buffer;Tris-Cl, phosphate buffered saline; Tris buffered saline; boratebuffered saline; and the like. An example is 1×Tris-Cl buffer, pH 8.8,0.1% SDS, room temperature. An exemplary assay for determining whether aDsRed mutant of the invention forms aggregates is as described in theExamples. In brief, a standard sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SOS-PAGE) protocol is used to separate DsRed mutantproteins produced recombinantly in a bacterial cell, e.g., E. coli.Samples are not boiled before loading onto the gel. Standard conditionsfor SDS-PAGE are described in Short Protocols in Molecular Biology,4^(th) Ed. 1999, FM Ausubel et al., eds., John Wiley & Sons, Inc.Typically, samples are electrophoresed in the presence of about 0.1% SDSin 1×Tris-Cl buffer (pH about 8.8).

In some embodiments, a subject non-aggregating polypeptide exhibitsreduced aggregation in vivo. “Reduced aggregation in vivo” refers toreduced aggregation in a cell. In some embodiments, the non-aggregatingpolypeptide shows less than about 90%, less than about 80%, less thanabout 70%, less than about 60%, less than about 50%, less than about40%, less than about 30%, less than about 25%, less than about 20%, lessthan about 15%, less than about 10%, or less than about 5% of theaggregation shown by its corresponding aggregating analogue under thesame in vivo conditions, e.g., in another eukaryotic cell from the samecell line, in an identical prokaryotic cell, or in a eukaryotic cell orcell population of the same cell type. In general, less than about 60%,less than about 50%, less than about 40%, less than about 30%, less thanabout 20%, less than about 10%, or less than about 5%, of the subjectnon-aggregating polypeptide present in a cell or a cell population isaggregated. Methods of measuring the degree of aggregation are known inthe art; any known method can be used to determine whether a givenmutant shows a reduction in aggregation compared to correspondingaggregating analogue, e.g., when compared to a corresponding aggregatingwild type polypeptide. Such methods include, but are not limited to,“pseudo-native” protein gel electrophoresis, as described in theExamples; gel filtration; ultracentrifugation; circular dichroism; andlight scattering. Aggregation can be measured by light scattering, asdescribed in the Examples. For non-aggregated proteins, the ratio ofabsorption at a shorter wavelength to the absorption at a longerwavelength is close to zero. In some embodiments, the ratio ofabsorption at 400 nm to the absorption at 566 nm of a non-aggregatingpolypeptide is in the range of from about 0.01 to about 0.1, from about0.015 to about 0.09, from about 0.02 to about 0.08, from about 0.025 toabout 0.07, or from about 0.03 to about 0.06.

In many embodiments, the non-aggregating polypeptides of the presentinvention have amino acid sequences that differ from their correspondingwild type sequences by a mutation in the N-terminus that modulates thecharges appearing on side groups of the N-terminus residues, e.g., toreverse or neutralize the charge, in a manner sufficient to produce anon-aggregating mutant of the naturally occurring protein or aggregatingmutant thereof. More specifically, basic residues located near theN-termini of the proteins are substituted, e.g., Lys and Arg residuesclose to the N-terminus are substituted with negatively charged orneutral residues. By N-terminus is meant within about 50 residues fromthe N-terminus, often within about 25 residues of the N-terminus andmore often within about 15 residues of the N-terminus, where in manyembodiments, residue modifications occur within about 10 residues of theN-terminus. Specific residues of interest in many embodiments include:2, 3, 4, 5, 6, 7, 8, 9 and 10.

As mentioned above, in addition to the non-aggregating feature of thesubject polypeptides encoded by the subject nucleic acids, the subjectpolypeptides are also characterized in that they are colored and/orfluorescence. By chromo and/or fluorescent protein is meant a proteinthat is colored, i.e., is pigmented, where the protein may or may not befluorescent, e.g., it may exhibit low, medium or high fluorescence uponirradiation with light of an excitation wavelength. In any event, thesubject proteins of interest are those in which the coloredcharacteristic, i.e., the chromo and/or fluorescent characteristic, isone that arises from the interaction of two or more residues of theprotein, and not from a single residue, more specifically a single sidechain of a single residue, of the protein. As such, fluorescent proteinsof the subject invention do not include proteins that exhibitfluorescence only from residues that act by themselves as intrinsicfluors, i.e., tryptophan, tyrosine and phenylalanine. As such, thefluorescent proteins of the subject invention are fluorescent proteinswhose fluorescence arises from some structure in the protein that isother than the above specified single residues, e.g., it arises from aninteraction of two or more residues.

In many embodiments, the polypeptides encoded by the subject nucleicacids are mutants of naturally occurring proteins, often proteins thatoccur in Cnidarian species, e.g., Anthozoan species. In certainembodiments, the nucleic acids are further characterized in that theyencode non-aggregating mutants of wild type proteins (or mutantsthereof) that are either from: (1) non-bioluminescent species, oftennon-bioluminescent Cnidarian species, e.g., non-bioluminescent Anthozoanspecies; or (2) from Anthozoan species that are not Pennatulaceanspecies, i.e., that are not sea pens. As such, the nucleic acids ofthese embodiments may encode non-aggregating mutants of proteins frombioluminescent Anthozoan species, so long as these species are notPennatulacean species, e.g., that are not Renillan or Ptilosarcanspecies. Of particular interest in certain embodiments arenon-aggregating mutants of the following specific wild type proteins (ormutants thereof): (1) amFP485, cFP484, zFP506, zFP540, drFP585, dsFP484,asFP600, dgFP512, dmFP592, as disclosed in application Ser. No.10/006,922, the disclosure of which is herein incorporated by reference;(2) hcFP640, as disclosed in application Ser. No. 09/976,673, thedisclosure of which is herein incorporated by reference; (3) CgCP, asdisclosed in application Ser. No. 60/255,533, the disclosure of which isherein incorporated by reference; and (4) hcriGFP, zoanRFP, scubGFP1,scubGFP2, rfloRFP, rfloGFP, mcavRFP, mcavGFP, cgigGFP, afraGFP,rfloGFP2, mcavGFP2, mannFP, as disclosed in application Ser. No.60/332,980, the disclosure of which is herein incorporated by reference.Specific non-aggregating fluorescent polypeptides of interest include,but are not limited to: FP1-NA; FP3-NA; FP4-NA; FP6-NA; E5-NA; 6/9Q-NA;7A-NA; mutM35-5 dimer-NA; and the like, where these particularnon-aggregating mutants are further described infra.

In some embodiments, the subject nucleic acids encode a polypeptidethat, in addition to the above characteristics, exhibits an increasedisoelectric point (pI) relative to a reference protein, e.g., acorresponding wild type protein. Isoelectric point can be determinedusing any method known in the art. In some embodiments, the pI is atheoretical pI, calculated as described in the Examples. In someembodiments, a mutant protein of the invention has a pI in the range offrom about 5.50 to about 7.00, from about 5.75 to about 6.75, from about6.00 to about 6.50, or from about 6.10 to about 6.40.

Fluorescence brightness of a particular fluorescent protein isdetermined by its quantum yield multiplied by maximal extinctioncoefficient. Brightness of a chromoprotein may be expressed by itsmaximal extinction coefficient. In some embodiments, the subject nucleicacids encoded polypeptides show substantially the same or greaterbrightness in a cell than a reference protein, e.g., compared to thecorresponding wild type protein, e.g., a mutant may be at least about10%, at least about 20%, at least about 30%, at least about 40%, atleast about 50%, at least about 60%, at least about 70%, at least about80%, at least about 90%, at least about 100% (or two-fold), at leastabout 150%, at least about three-fold, or at least about four-fold, ormore, brighter in a cell than the reference protein. A “cell” can be aprokaryotic cell or a eukaryotic cell. Methods of measuring brightnessare known in the art. Brightness can be measured using any known method,including, but not limited to, visual screening, spectrophotometry,spectrofluorometry, fluorescent microscopy, by fluorescence activatedcell sorting (FACS) machines, etc. In some instances, brightness of asubject mutant protein in a cell can be visually compared to thebrightness of a reference protein in a cell of the same cell type, oranother cell of the same cell line.

By nucleic acid composition is meant a composition comprising a sequenceof DNA having an open reading frame that encodes a non-aggregatingchromo/fluoro polypeptide of the subject invention, i.e., achromo/fluoroprotein coding sequence, and is capable, under appropriateconditions, of being expressed as a non-aggregating chromo/fluoroprotein according to the subject invention. Also encompassed in thisterm are nucleic acids that are homologous, substantially similar oridentical to the nucleic acids of the present invention. Thus, thesubject invention provides coding sequences encoding the proteins of thesubject invention, as well as homologs thereof.

In addition to the above described specific nucleic acid compositions,also of interest are homologues of the above sequences. In certainembodiments, sequence similarity between homologues is at least about20%, sometimes at least about 25%, and may be 30%, 35%, 40%, 50%, 60%,70% or higher, including 75%, 80%, 85%, 90% and 95% or higher. Sequencesimilarity is calculated based on a reference sequence, which may be asubset of a larger sequence, such as a conserved motif, coding region,flanking region, etc. A reference sequence will usually be at leastabout 18 nt long, more usually at least about 30 nt long, and may extendto the complete sequence that is being compared. Algorithms for sequenceanalysis are known in the art, such as BLAST, described in Altschul etal., (1990), J. Mol. Biol. 215:403-10 (using default settings, i.e.parameters w=4 and T=17). Of particular interest in certain embodimentsare nucleic acids of substantially the same length as the nucleic acididentified as SEQ ID NOS: 13; 14; 15; 17; 19; 21; and 23, where bysubstantially the same length is meant that any difference in lengthdoes not exceed about 20 number %, usually does not exceed about 10number % and more usually does not exceed about 5 number %; and havesequence identity to any of these sequences of at least about 90%,usually at least about 95% and more usually at least about 99% over theentire length of the nucleic acid. In many embodiments, the nucleicacids have a sequence that is substantially similar (i.e. the same as)or identical to the sequences of SEQ ID NOS: 14; 15; 17; 19; 21; and 23.By substantially similar is meant that sequence identity will generallybe at least about 60%, usually at least about 75% and often at leastabout 80, 85, 90, or even 95%.

Also provided are nucleic acids that encode the proteins encoded by theabove described nucleic acids, but differ in sequence from the abovedescribed nucleic acids due to the degeneracy of the genetic code.

Also provided are nucleic acids that hybridize to the above describednucleic acids under stringent conditions. An example of stringenthybridization conditions is hybridization at 50° C. or higher and0.1×SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another exampleof stringent hybridization conditions is overnight incubation at 42° C.in a solution: 50% formamide, 5×SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH7.6), 5×Denhardt's solution, 10%dextran sulfate, and 20 μg/ml denatured, sheared salmon sperm DNA,followed by washing the filters in 0.1×SSC at about 65° C. Stringenthybridization conditions are hybridization conditions that are at leastas stringent as the above representative conditions, where conditionsare considered to be at least as stringent if they are at least about80% as stringent, typically at least about 90% as stringent as the abovespecific stringent conditions. Other stringent hybridization conditionsare known in the art and may also be employed to identify nucleic acidsof this particular embodiment of the invention.

Nucleic acids encoding mutants of the non-aggregating proteins of theinvention are also provided. Mutant nucleic acids can be generated byrandom mutagenesis or targeted mutagenesis, using well-known techniqueswhich are routine in the art. In some embodiments, chromo- orfluorescent proteins encoded by nucleic acids encoding homologues ormutants have the same fluorescent properties as the wild-typefluorescent protein. In other embodiments, homologue or mutant nucleicacids encode chromo- or fluorescent proteins with altered spectralproperties, as described in more detail herein.

The subject nucleic acids may be present in an appropriate vector forextrachromosomal maintenance or for integration into a host genome, asdescribed in greater detail below.

The nucleic acid compositions of the subject invention may encode all ora part of the subject proteins. Double or single stranded fragments maybe obtained from the DNA sequence by chemically synthesizingoligonucleotides in accordance with conventional methods, by restrictionenzyme digestion, by PCR amplification, etc. For the most part, DNAfragments will be of at least about 15 nt, usually at least about 18 ntor about 25 nt, and may be at least about 50 nt. In some embodiments,the subject nucleic acid molecules may be about 100 nt, about 200 nt,about 300 nt, about 400 nt, about 500 nt, about 600 nt, about 700 nt, orabout 720 nt in length. The subject nucleic acids may encode fragmentsof the subject proteins or the full-length proteins, e.g., the subjectnucleic acids may encode polypeptides of about 25 aa, about 50 aa, about75 aa, about 100 aa, about 125 aa, about 150 aa, about 200 aa, about 210aa, about 220 aa, about 230 aa, or about 240 aa, up to the entireprotein.

The subject polynucleotides and constructs thereof are provided. Thesemolecules can be generated synthetically by a number of differentprotocols known to those of skill in the art. Appropriate polynucleotideconstructs are purified using standard recombinant DNA techniques asdescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, 2nd Ed., (1989) Cold Spring Harbor Press, Cold SpringHarbor, N.Y., and under current regulations described in United StatesDept. of HHS, National Institute of Health (NIH) Guidelines forRecombinant DNA Research.

Also provided are nucleic acids that encode fusion proteins of thesubject proteins, or fragments thereof, which are fused to a secondprotein, e.g., a degradation sequence, a signal peptide, a protein ofinterest (i.e., a protein being studied), etc. Fusion proteins maycomprise a subject polypeptide, or fragment thereof, and anon-non-aggregating polypeptide (“the fusion partner”) fused in-frame atthe N-terminus and/or C-terminus of the subject polypeptide. Fusionpartners include, but are not limited to, polypeptides that can bindantibody specific to the fusion partner (e.g., epitope tags); antibodiesor binding fragments thereof; polypeptides that provide a catalyticfunction or induce a cellular response; ligands or receptors or mimeticsthereof; and the like. In such fusion proteins, the fusion partner isgenerally not naturally associated with the subject non-aggregatingprotein portion of the fusion protein, and in certain embodiments not anCnidarian protein or derivative/fragment thereof, i.e., it is not foundin Cnidarian species.

Also provided are constructs comprising the subject nucleic acidsinserted into a vector, where such constructs may be used for a numberof different applications, including propagation, protein production,etc. Viral and non-viral vectors may be prepared and used, includingplasmids. The choice of vector will depend on the type of cell in whichpropagation is desired and the purpose of propagation. Certain vectorsare useful for amplifying and making large amounts of the desired DNAsequence. Other vectors are suitable for expression in cells in culture.Still other vectors are suitable for transfer and expression in cells ina whole animal or person. The choice of appropriate vector is wellwithin the skill of the art. Many such vectors are availablecommercially. To prepare the constructs, the partial or full-lengthpolynucleotide is inserted into a vector typically by means of DNAligase attachment to a cleaved restriction enzyme site in the vector.Alternatively, the desired nucleotide sequence can be inserted byhomologous recombination in vivo. Typically this is accomplished byattaching regions of homology to the vector on the flanks of the desirednucleotide sequence. Regions of homology are added by ligation ofoligonucleotides, or by polymerase chain reaction using primerscomprising both the region of homology and a portion of the desirednucleotide sequence, for example.

Also provided are expression cassettes or systems that find use in,among other applications, the synthesis of the subject proteins. Forexpression, the gene product encoded by a polynucleotide of theinvention is expressed in any convenient expression system, including,for example, bacterial, yeast, insect, amphibian and mammalian systems.Suitable vectors and host cells are described in U.S. Pat. No.5,654,173. In the expression vector, a subject polynucleotide is linkedto a regulatory sequence as appropriate to obtain the desired expressionproperties. These regulatory sequences can include promoters (attachedeither at the 5′ end of the sense strand or at the 3′ end of theantisense strand), enhancers, terminators, operators, repressors, andinducers. The promoters can be regulated or constitutive. In somesituations it may be desirable to use conditionally active promoters,such as tissue-specific or developmental stage-specific promoters. Theseare linked to the desired nucleotide sequence using the techniquesdescribed above for linkage to vectors. Any techniques known in the artcan be used. In other words, the expression vector will provide atranscriptional and translational initiation region, which may beinducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the subject species from whichthe subject nucleic acid is obtained, or may be derived from exogenoussources.

Expression vectors generally have convenient restriction sites locatednear the promoter sequence to provide for the insertion of nucleic acidsequences encoding heterologous proteins. A selectable marker operativein the expression host may be present. Expression vectors may be usedfor, among other things, the production of fusion proteins, as describedabove.

Expression cassettes may be prepared comprising a transcriptioninitiation region, the gene or fragment thereof, and a transcriptionaltermination region. Of particular interest is the use of sequences thatallow for the expression of functional epitopes or domains, usually atleast about 8 amino acids in length, more usually at least about 15amino acids in length, to about 25 amino acids, and up to the completeopen reading frame of the gene. After introduction of the DNA, the cellscontaining the construct may be selected by means of a selectablemarker, the cells expanded and then used for expression.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. For large scale production of the protein, aunicellular organism, such as E. coli, B. subtilis, S. cerevisiae,insect cells in combination with baculovirus vectors, or cells of ahigher organism such as vertebrates, e.g. COS 7 cells, HEK 293, CHO,Xenopus Oocytes, etc., may be used as the expression host cells. In somesituations, it is desirable to express the gene in eukaryotic cells,where the expressed protein will benefit from native folding andpost-translational modifications. Small peptides can also be synthesizedin the laboratory. Polypeptides that are subsets of the complete proteinsequence may be used to identify and investigate parts of the proteinimportant for function.

Specific expression systems of interest include bacterial, yeast, insectcell and mammalian cell derived expression systems. Representativesystems from each of these categories is are provided below:

Bacteria. Expression systems in bacteria include those described inChang et al., Nature (1978) 275:615; Goeddel et al., Nature (1979)281:544; Goeddel et al., Nucleic Acids Res. (1980) 8:4057; EP 0 036,776;U.S. Pat. No. 4,551,433; DeBoer et al., Proc. Natl. Acad. Sci. (USA)(1983) 80:21-25; and Siebenlist et al., Cell (1980) 20:269.

Yeast. Expression systems in yeast include those described in Hinnen etal., Proc. Natl. Acad. Sci. (USA) (1978) 75:1929; Ito et al., J.Bacterial. (1983) 153:163; Kurtz et al., Mol. Cell. Biol. (1986) 6:142;Kunze et al., J. Basic Microbiol. (1985) 25:141; Gleeson et al., J. Gen.Microbiol. (1986) 132:3459; Roggenkamp et al., Mol. Gen. Genet. (1986)202:302; Das et al., J. Bacterial. (1984) 158:1165; De Louvencourt etal., J. Bacteriol. (1983) 154:737; Van den Berg et al., Bio/Technology(1990) 8:135; Kunze et al., J. Basic Microbiol. (1985) 25:141; Gregg etal., Mol. Cell. Biol. (1985) 5:3376; U.S. Pat. Nos. 4,837,148 and4,929,555; Beach and Nurse, Nature (1981) 300:706; Davidow et al., Curr.Genet. (1985) 10:380; Gaillardin et al., Curr. Genet. (1985) 10:49;Ballance et al., Biochem. Biophys. Res. Commun. (1983) 112:284-289;Tilburn et al., Gene (1983) 26:205-221; Yelton et al., Proc. Natl. Acad.Sci. (USA) (1984) 81:1470-1474; Kelly and Hynes, EMBO J. (1985)4:475479; EP 0 244,234; and WO 91/00357.

Insect Cells. Expression of heterologous genes in insects isaccomplished as described in U.S. Pat. No. 4,745,051; Friesen et al.,“The Regulation of Baculovirus Gene Expression”, in: The MolecularBiology Of Baculoviruses (1986) (W. Doerfler, ed.); EP 0 127,839; EP 0155,476; and Vlak et al., J. Gen. Virol. (1988) 69:765-776; Miller atal., Ann. Rev. Microbial. (1988) 42:177; Carbonell et al., Gene (1988)73:409; Maeda at al., Nature (1985) 315:592-594; Lebacq-Verheyden etal., Mol. Cell. Biol. (1988) 8:3129; Smith et al., Proc. Natl. Acad.Sci. (USA) (1985) 82:8844; Miyajima at al., Gene (1987) 58:273; andMartin et al., DNA (1988) 7:99. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts aredescribed in Luckow et al., Bio/Technology (1988) 6:47-55, Miller etal., Generic Engineering (1986) 8:277-279, and Maeda et al., Nature(1985) 315:592-594.

Mammalian Cells. Mammalian expression is accomplished as described inDijkema et al., EMBO J. (1985) 4:761, Gorman et al., Proc. Natl. Acad.Sci. (USA) (1982) 79:6777, Boshart et al., Cell (1985) 41:521 and U.S.Pat. No. 4,399,216. Other features of mammalian expression arefacilitated as described in Ham and Wallace, Meth. Enz. (1979) 58:44,Barnes and Sato, Anal. Biochem. (1980) 102:255, U.S. Pat. Nos.4,767,704, 4,657,866, 4,927,762, 4,560,655, WO 90/103430, WO 87/00195,and U.S. RE 30,985.

When any of the above host cells, or other appropriate host cells ororganisms, are used to replicate and/or express the polynucleotides ornucleic acids of the invention, the resulting replicated nucleic acid,RNA, expressed protein or polypeptide, is within the scope of theinvention as a product of the host cell or organism. The product isrecovered by any appropriate means known in the art.

The subject nucleic acids may be mutated in various ways known in theart to generate targeted changes in the sequence of the encoded protein,properties of the encoded protein, including fluorescent properties ofthe encoded protein, etc. The DNA sequence or protein product of such amutation will usually be substantially similar to the sequences providedherein, e.g. will differ by at least one nucleotide or amino acid,respectively, and may differ by at least two but not more than about tennucleotides or amino acids. The sequence changes may be substitutions,insertions, deletions, or a combination thereof. Deletions may furtherinclude larger changes, such as deletions of a domain or exon, e.g. ofstretches of 10, 20, 50, 75, 100, 150 or more as residues. Techniquesfor in vitro mutagenesis of cloned genes are known. Examples ofprotocols for site specific mutagenesis may be found in Gustin et al.(1993), Biotechniques 14:22; Barany (1985), Gene 37:111-23; Colicelli etal. (1985), Mol. Gen. Genet. 199:537-9; and Prentki et al. (1984), Gene29:303-13. Methods for site specific mutagenesis can be found inSambrook et al., Molecular Cloning: A Laboratory Manual, CSH Press 1989,pp. 15.3-15.108; Weiner et al. (1993), Gene 126:35-41; Sayers at al.(1992), Biotechniques 13:592-6; Jones and Winistorfer (1992),Biotechniques 12:528-30; Barton et al. (1990), Nucleic Acids Res18:7349-55; Marotti and Tomich (1989), Gene Anal. Tech. 6:67-70; and Zhu(1989), Anal Biochem 177:120-4. Such mutated nucleic acid derivativesmay be used to study structure-function relationships of a particularchromo/fluorescent protein, or to alter properties of the protein thataffect its function or regulation.

Also of interest are humanized versions of the subject nucleic acids. Asused herein, the term “humanized” refers to changes made to the nucleicacid sequence to optimize the codons for expression of the protein inhuman cells (Yang et al., Nucleic Acids Research 24 (1996), 4592-4593).See also U.S. Pat. No. 5,795,737 which describes humanization ofproteins, the disclosure of which is herein incorporated by reference.

Protein/Polypeptide Compositions

Also provided by the subject invention are non-aggregating chromo-and/or fluorescent proteins and mutants thereof encoded by the subjectnucleic acids, as well as polypeptide compositions related thereto. Theterm polypeptide composition as used herein refers to both thefull-length protein, as well as portions or fragments thereof. Alsoincluded in this term are variations of the naturally occurring protein,where such variations are homologous or substantially similar to thenaturally occurring protein, and mutants of the naturally occurringproteins, as described in greater detail below.

In many embodiments, the subject proteins have an absorbance maximumranging from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm. Where the subject proteins arefluorescent proteins, by which is meant that they can be excited at onewavelength of light following which they will emit light at anotherwavelength, the excitation spectra of the subject proteins typicallyranges from about 300 to 700, usually from about 350 to 650 and moreusually from about 400 to 600 nm while the emission spectra of thesubject proteins typically ranges from about 400 to 800, usually fromabout 425 to 775 and more usually from about 450 to 750 nm. The subjectproteins generally have a maximum extinction coefficient that rangesfrom about 10,000 to 50,000 and usually from about 15,000 to 45,000. Thesubject proteins typically range in length from about 150 to 300 andusually from about 200 to 300 amino acid residues, and generally have amolecular weight ranging from about 15 to 35 kDa, usually from about17.5 to 32.5 kDa.

In certain embodiments, the subject proteins are bright, where by brightis meant that the chromoproteins and their fluorescent mutants can bedetected by common methods (e.g., visual screening, spectrophotometry,spectrofluorometry, fluorescent microscopy, by FACS machines, etc.)Fluorescence brightness of particular fluorescent proteins is determinedby its quantum yield multiplied by maximal extinction coefficient.Brightness of a chromoproteins may be expressed by its maximalextinction coefficient.

In certain embodiments, the subject proteins fold rapidly followingexpression in the host cell. By rapidly folding is meant that theproteins achieve their tertiary structure that gives rise to theirchromo- or fluorescent quality in a short period of time. In theseembodiments, the proteins fold in a period of time that generally doesnot exceed about 3 days, usually does not exceed about 2 days and moreusually does not exceed about 1 day.

Specific proteins of interest are non-aggregating mutant polypeptides orvariants of chromo/fluoroproteins (and mutants thereof) from thefollowing specific Anthozoan species: Anemonia majano, Clavularia sp.,Zoanthus sp., Zoanthus sp., Discosoma striata, Discosoma sp. “red”,Anemonia sulcata, Discosoma sp “green”, Discosoma sp. “magenta.”Specific non-aggregating fluorescent polypeptides of interest include,but are not limited to: FP1-NA; FP3-NA; FP4-NA; FP6-NA; E5-NA; 6/9Q-NA;7A-NA; and the like.

Homologs or proteins (or fragments thereof) that vary in sequence fromthe amino acid sequences of the above provided specific non-aggregatingpolypeptides are also provided. By homolog is meant a protein having atleast about 10%, usually at least about 20% and more usually at leastabout 30%, and in many embodiments at least about 35%, usually at leastabout 40% and more usually at least about 60% amino acid sequenceidentity to the protein of the subject invention, as determined usingMegAlign, DNAstar (1998) clustal algorithm as described in D. G. Higginsand P. M. Sharp, “Fast and Sensitive multiple Sequence Alignments on aMicrocomputer,” (1989) CABIOS, 5: 151-153. (Parameters used are ktuple1, gap penalty 3, window, 5 and diagonals saved 5). In many embodiments,homologues of interest have much higher sequence identify, e.g., 65%,70%, 75%, 80%, 85%, 90% or higher.

Also provided are proteins that are substantially identical to thesequences of the above provided specific proteins, where bysubstantially identical is meant that the protein has an amino acidsequence identity to the one of the above specifically provided proteinsof at least about 60%, usually at least about 65% and more usually atleast about 70%, where in some instances the identity may be muchhigher, e.g., 75%, 80%, 85%, 90%, 95% or higher.

In many embodiments, the subject homologues have structural featuresfound in the above provided specific sequences, where such structuralfeatures include the β-can fold.

Proteins which are mutants of the above specifically described proteinsare also provided. Mutants may retain biological properties of thewild-type (e.g., naturally occurring) proteins, or may have biologicalproperties which differ from the wild-type proteins. The term“biological property” of the subject proteins includes, but is notlimited to, spectral properties, such as absorbance maximum, emissionmaximum, maximum extinction coefficient, brightness (e.g., as comparedto the wild-type protein or another reference protein such as greenfluorescent protein from A. victoria), and the like; in vivo and/or invitro stability (e.g., half-life); etc. Mutants include single aminoacid changes, deletions of one or more amino acids, N-terminaltruncations, C-terminal truncations, insertions, etc.

Mutants can be generated using standard techniques of molecular biology,e.g., random mutagenesis, and targeted mutagenesis. Several mutants aredescribed herein. Given the guidance provided in the Examples, and usingstandard techniques, those skilled in the art can readily generate awide variety of additional mutants and test whether a biologicalproperty has been altered. For example, fluorescence intensity can bemeasured using a spectrophotometer at various excitation wavelengths.

Mutants of the above specifically provided proteins are also provided.Generally such polypeptides include an amino acid sequence encoded by anopen reading frame (ORF) of the gene encoding the subject wild typeprotein, including the full length protein and fragments thereof,particularly biologically active fragments and/or fragmentscorresponding to functional domains, and the like; and including fusionsof the subject polypeptides to other proteins or parts thereof.Fragments of interest will typically be at least about 10 aa in length,usually at least about 50 aa in length, and may be as long as 300 aa inlength or longer, but will usually not exceed about 1000 aa in length,where the fragment will have a stretch of amino acids that is identicalto the subject protein of at least about 10 aa, and usually at leastabout 15 aa, and in many embodiments at least about 50 aa in length. Insome embodiments, the subject polypeptides are about 25 aa, about 50 aa,about 75 aa, about 100 aa, about 125 aa, about 150 aa, about 200 aa,about 210 aa, about 220 aa, about 230 aa, or about 240 aa in length, upto the entire protein. In some embodiments, a protein fragment retainsall or substantially all of a biological property of the wild-typeprotein.

The subject proteins and polypeptides may be synthetically producedusing any convenient protocol, e.g., by expressing a recombinant gene ornucleic acid coding sequence encoding the protein of interest in asuitable host, as described above. Any convenient protein purificationprocedures may be employed, where suitable protein purificationmethodologies are described in Guide to Protein Purification, (Deuthsered.) (Academic Press, 1990). For example, a lysate may prepared from theoriginal source and purified using HPLC, exclusion chromatography, gelelectrophoresis, affinity chromatography, and the like.

Antibody Compositions

Also provided are antibodies that specifically bind to the subjectnon-aggregating fluorescent proteins. Suitable antibodies are obtainedby immunizing a host animal with peptides comprising all or a portion ofthe subject protein. Suitable host animals include mouse, rat sheep,goat, hamster, rabbit, etc. The immunogen may comprise the completeprotein, or fragments and derivatives thereof.

For preparation of polyclonal antibodies, the first step is immunizationof the host animal with the target protein, where the target proteinwill preferably be in substantially pure form, comprising less thanabout 1% contaminant. The immunogen may comprise the complete targetprotein, fragments or derivatives thereof. To increase the immuneresponse of the host animal, the target protein may be combined with anadjuvant, where suitable adjuvants include alum, dextran, sulfate, largepolymeric anions, oil & water emulsions, e.g. Freund's adjuvant,Freund's complete adjuvant, and the like. The target protein may also beconjugated to synthetic carrier proteins or synthetic antigens. Avariety of hosts may be immunized to produce the polyclonal antibodies.Such hosts include rabbits, guinea pigs, rodents, e.g. mice, rats,sheep, goats, and the like. The target protein is administered to thehost, usually intradermally, with an initial dosage followed by one ormore, usually at least two, additional booster dosages. Followingimmunization, the blood from the host will be collected, followed byseparation of the serum from the blood cells. The Ig present in theresultant antiserum may be further fractionated using known methods,such as ammonium salt fractionation, DEAE chromatography, and the like.

Monoclonal antibodies are produced by conventional techniques.Generally, the spleen and/or lymph nodes of an immunized host animalprovide a source of plasma cells. The plasma cells are immortalized byfusion with myeloma cells to produce hybridoma cells. Culturesupernatant from individual hybridomas is screened using standardtechniques to identify those producing antibodies with the desiredspecificity. Suitable animals for production of monoclonal antibodies tothe human protein include mouse, rat, hamster, etc. To raise antibodiesagainst the mouse protein, the animal will generally be a hamster,guinea pig, rabbit, etc. The antibody may be purified from the hybridomacell supernatants or ascites fluid by conventional techniques, e.g.affinity chromatography using protein bound to an insoluble support,protein A sepharose, etc.

The antibody may be produced as a single chain, instead of the normalmultimeric structure. Single chain antibodies are described in Jost etal. (1994) J.B.C. 269:26267-73, and others. DNA sequences encoding thevariable region of the heavy chain and the variable region of the lightchain are ligated to a spacer encoding at least about 4 amino acids ofsmall neutral amino acids, including glycine and/or serine. The proteinencoded by this fusion allows assembly of a functional variable regionthat retains the specificity and affinity of the original antibody.

Also of interest in certain embodiments are humanized antibodies.Methods of humanizing antibodies are known in the art. The humanizedantibody may be the product of an animal having transgenic humanimmunoglobulin constant region genes (see for example internationalPatent Applications WO 90/10077 and WO 90/04036). Alternatively, theantibody of interest may be engineered by recombinant DNA techniques tosubstitute the CH1, CH2, CH3, hinge domains, and/or the framework domainwith the corresponding human sequence (see WO 92/02190).

The use of Ig cDNA for construction of chimeric immunoglobulin genes isknown in the art (Liu et al. (1987) P.N.A.S. 84:3439 and (1987) J.Immunol. 139:3521). mRNA is isolated from a hybridoma or other cellproducing the antibody and used to produce cDNA. The cDNA of interestmay be amplified by the polymerase chain reaction using specific primers(U.S. Pat. Nos. 4,683,195 and 4,683,202). Alternatively, a library ismade and screened to isolate the sequence of interest. The DNA sequenceencoding the variable region of the antibody is then fused to humanconstant region sequences. The sequences of human constant regions genesmay be found in Kabat et al. (1991) Sequences of Proteins ofImmunological Interest, N.I.H. publication no. 91-3242. Human C regiongenes are readily available from known clones. The choice of isotypewill be guided by the desired effector functions, such as complementfixation, or activity in antibody-dependent cellular cytotoxicity.Preferred isotypes are IgG1, IgG3 and IgG4. Either of the human lightchain constant regions, kappa or lambda, may be used. The chimeric,humanized antibody is then expressed by conventional methods.

Antibody fragments, such as Fv, F(ab′)₂ and Fab may be prepared bycleavage of the intact protein, e.g. by protease or chemical cleavage.Alternatively, a truncated gene is designed. For example, a chimericgene encoding a portion of the F(ab′)₂ fragment would include DNAsequences encoding the CH1 domain and hinge region of the H chain,followed by a translational stop codon to yield the truncated molecule.

Consensus sequences of H and L J regions may be used to designoligonucleotides for use as primers to introduce useful restrictionsites into the J region for subsequent linkage of V region segments tohuman C region segments. C region cDNA can be modified by site directedmutagenesis to place a restriction site at the analogous position in thehuman sequence.

Expression vectors include plasmids, retroviruses, YACs, EBV derivedepisomes, and the like. A convenient vector is one that encodes afunctionally complete human CH or CL immunoglobulin sequence, withappropriate restriction sites engineered so that any VH or VL sequencecan be easily inserted and expressed. In such vectors, splicing usuallyoccurs between the splice donor site in the inserted J region and thesplice acceptor site preceding the human C region, and also at thesplice regions that occur within the human CH exons. Polyadenylation andtranscription termination occur at native chromosomal sites downstreamof the coding regions. The resulting chimeric antibody may be joined toany strong promoter, including retroviral LTRs, e.g. SV-40 earlypromoter, (Okayama et al. (1983) Mol. Cell. Bio. 3:280), Rous sarcomavirus LTR (Gorman et al. (1982) P.N.A.S. 79:6777), and moloney murineleukemia virus LTR (Grosschedl et al. (1985) Cell 41:885); native Igpromoters, etc.

Transgenics

The subject nucleic acids can be used to generate transgenic, non-humanplants or animals or site specific gene modifications in cell lines.Transgenic cells of the subject invention include one or more nucleicacids according to the subject invention present as a transgene, whereincluded within this definition are the parent cells transformed toinclude the transgene and the progeny thereof. In many embodiments, thetransgenic cells are cells that do not normally harbor or contain anucleic acid according to the subject invention. In those embodimentswhere the transgenic cells do naturally contain the subject nucleicacids, the nucleic acid will be present in the cell in a position otherthan its natural location, i.e. integrated into the genomic material ofthe cell at a non-natural location. Transgenic animals may be madethrough homologous recombination, where the endogenous locus is altered.Alternatively, a nucleic acid construct is randomly integrated into thegenome. Vectors for stable integration include plasmids, retrovirusesand other animal viruses, YACs, and the like.

Transgenic organisms of the subject invention include cells andmulticellular organisms, e.g., plants and animals, that are endogenousknockouts in which expression of the endogenous gene is at least reducedif not eliminated. Transgenic organisms of interest also include cellsand multicellular organisms, e.g., plants and animals, in which theprotein or variants thereof is expressed in cells or tissues where it isnot normally expressed and/or at levels not normally present in suchcells or tissues.

DNA constructs for homologous recombination will comprise at least aportion of the gene of the subject invention, wherein the gene has thedesired genetic modification(s), and includes regions of homology to thetarget locus. DNA constructs for random integration need not includeregions of homology to mediate recombination. Conveniently, markers forpositive and negative selection are included. Methods for generatingcells having targeted gene modifications through homologousrecombination are known in the art. For various techniques fortransfecting mammalian cells, see Keown et al., (1990), Meth. Enzymol.185:527-537.

For embryonic stem (ES) cells, an ES cell line may be employed, orembryonic cells may be obtained freshly from a host, e.g. mouse, rat,guinea pig, etc. Such cells are grown on an appropriatefibroblast-feeder layer or grown in the presence of leukemia inhibitingfactor (LIF). When ES or embryonic cells have been transformed, they maybe used to produce transgenic animals. After transformation, the cellsare plated onto a feeder layer in an appropriate medium. Cellscontaining the construct may be detected by employing a selectivemedium. After sufficient time for colonies to grow, they are picked andanalyzed for the occurrence of homologous recombination or integrationof the construct. Those colonies that are positive may then be used forembryo manipulation and blastocyst injection. Blastocysts are obtainedfrom 4 to 6 week old superovulated females. The ES cells aretrypsinized, and the modified cells are injected into the blastocoel ofthe blastocyst. After injection, the blastocysts are returned to eachuterine horn of pseudopregnant females. Females are then allowed to goto term and the resulting offspring screened for the construct. Byproviding for a different phenotype of the blastocyst and thegenetically modified cells, chimeric progeny can be readily detected.

The chimeric animals are screened for the presence of the modified geneand males and females having the modification are mated to producehomozygous progeny. If the gene alterations cause lethality at somepoint in development, tissues or organs can be maintained as allogeneicor congenic grafts or transplants, or in in vitro culture. Thetransgenic animals may be any non-human mammal, such as laboratoryanimals, domestic animals, etc. The transgenic animals may be used infunctional studies, drug screening, etc. Representative examples of theuse of transgenic animals include those described infra.

Transgenic plants may be produced in a similar manner. Methods ofpreparing transgenic plant cells and plants are described in U.S. Pat.Nos. 5,767,367; 5,750,870; 5,739,409; 5,689,049; 5,689,045; 5,674,731;5,656,466; 5,633,155; 5,629,470; 5,595,896; 5,576,198; 5,538,879;5,484,956; the disclosures of which are herein incorporated byreference. Methods of producing transgenic plants are also reviewed inPlant Biochemistry and Molecular Biology (eds Lea & Leegood, John Wiley& Sons)(1993) pp 275-295. In brief, a suitable plant cell or tissue isharvested, depending on the nature of the plant species. As such, incertain instances, protoplasts will be isolated, where such protoplastsmay be isolated from a variety of different plant tissues, e.g. leaf,hypoctyl, root, etc. For protoplast isolation, the harvested cells areincubated in the presence of cellulases in order to remove the cellwall, where the exact incubation conditions vary depending on the typeof plant and/or tissue from which the cell is derived. The resultantprotoplasts are then separated from the resultant cellular debris bysieving and centrifugation. Instead of using protoplasts, embryogenicexplants comprising somatic cells may be used for preparation of thetransgenic host. Following cell or tissue harvesting, exogenous DNA ofinterest is introduced into the plant cells, where a variety ofdifferent techniques are available for such introduction. With isolatedprotoplasts, the opportunity arise for introduction via DNA-mediatedgene transfer protocols, including: incubation of the protoplasts withnaked DNA, e.g. plasmids, comprising the exogenous coding sequence ofinterest in the presence of polyvalent cations, e.g. PEG or PLO; andelectroporation of the protoplasts in the presence of naked DNAcomprising the exogenous sequence of interest. Protoplasts that havesuccessfully taken up the exogenous DNA are then selected, grown into acallus, and ultimately into a transgenic plant through contact with theappropriate amounts and ratios of stimulatory factors, e.g. auxins andcytokinins. With embryogenic explants, a convenient method ofintroducing the exogenous DNA in the target somatic cells is through theuse of particle acceleration or “gene-gun” protocols. The resultantexplants are then allowed to grow into chimera plants, cross-bred andtransgenic progeny are obtained. Instead of the naked DNA approachesdescribed above, another convenient method of producing transgenicplants is Agrobacterium mediated transformation. With Agrobacteriummediated transformation, co-integrative or binary vectors comprising theexogenous DNA are prepared and then introduced into an appropriateAgrobacterium strain, e.g. A. tumefaciens. The resultant bacteria arethen incubated with prepared protoplasts or tissue explants, e.g. leafdisks, and a callus is produced. The callus is then grown underselective conditions, selected and subjected to growth media to induceroot and shoot growth to ultimately produce a transgenic plant.

Utility

The subject non-aggregating chromoproteins and fluorescent mutantsthereof find use in a variety of different applications, where theapplications necessarily differ depending on whether the protein is achromoprotein or a fluorescent protein. Representative uses for each ofthese types of proteins will be described below, where the followingdescribed uses are merely representative and are in no way meant tolimit the use of the subject proteins to those described below.

Chromoproteins

The subject chromoproteins of the present invention find use in avariety of different applications. One application of interest is theuse of the subject proteins as coloring agents which are capable ofimparting color or pigment to a particular composition of matter. Ofparticular interest in certain embodiments are non-toxic chromoproteins.The subject chromoproteins may be incorporated into a variety ofdifferent compositions of matter, where representative compositions ofmatter include: food compositions, pharmaceuticals, cosmetics, livingorganisms, e.g., animals and plants, and the like. Where used as acoloring agent or pigment, a sufficient amount of the chromoprotein isincorporated into the composition of matter to impart the desired coloror pigment thereto. The chromoprotein may be incorporated into thecomposition of matter using any convenient protocol, where theparticular protocol employed will necessarily depend, at least in part,on the nature of the composition of matter to be colored. Protocols thatmay be employed include, but are not limited to: blending, diffusion,friction, spraying, injection, tattooing, and the like.

The chromoproteins may also find use as labels in analyte detectionassays, e.g., assays for biological analytes of interest. For example,the chromoproteins may be incorporated into adducts with analytespecific antibodies or binding fragments thereof and subsequentlyemployed in immunoassays for analytes of interest in a complex sample,as described in U.S. Pat. No. 4,302,536; the disclosure of which isherein incorporated by reference. Instead of antibodies or bindingfragments thereof, the subject chromoproteins or chromogenic fragmentsthereof may be conjugated to ligands that specifically bind to ananalyte of interest, or other moieties, growth factors, hormones, andthe like; as is readily apparent to those of skill in the art.

In yet other embodiments, the subject chromoproteins may be used asselectable markers in recombinant DNA applications, e.g., the productionof transgenic cells and organisms, as described above. As such, one canengineer a particular transgenic production protocol to employexpression of the subject chromoproteins as a selectable marker, eitherfor a successful or unsuccessful protocol. Thus, appearance of the colorof the subject chromoprotein in the phenotype of the transgenic organismproduced by a particular process can be used to indicate that theparticular organism successfully harbors the transgene of interest,often integrated in a manner that provides for expression of thetransgene in the organism. When used a selectable marker, a nucleic acidencoding for the subject chromoprotein can be employed in the transgenicgeneration process, where this process is described in greater detailsupra. Particular transgenic organisms of interest where the subjectproteins may be employed as selectable markers include transgenicplants, animals, bacteria, fungi, and the like.

In yet other embodiments, the chromoproteins (and fluorescent proteins)of the subject invention find use in sunscreens, as selective filters,etc., in a manner similar to the uses of the proteins described in WO00/46233.

Fluorescent Proteins

The subject fluorescent proteins of the present invention (as well asother components of the subject invention described above) find use in avariety of different applications, where such applications include, butare not limited to, the following. The first application of interest isthe use of the subject proteins in fluorescence resonance energytransfer (FRET) applications. In these applications, the subjectproteins serve as donor and/or acceptors in combination with a secondfluorescent protein or dye, e.g., a fluorescent protein as described inMatz et al., Nature Biotechnology (October 1999) 17:969-973, a greenfluorescent protein from Aequoria victoria or fluorescent mutantthereof, e.g., as described in U.S. Pat. Nos. 6,066,476; 6,020,192;5,985,577; 5,976,796; 5,968,750; 5,968,738; 5,958,713; 5,919,445;5,874,304, the disclosures of which are herein incorporated byreference, other fluorescent dyes, e.g., coumarin and its derivatives,e.g. 7-amino-4-methylcoumarin, aminocoumarin, bodipy dyes, such asBodipy FL, cascade blue, fluorescein and its derivatives, e.g.fluorescein isothiocyanate, Oregon green, rhodamine dyes, e.g. texasred, tetramethylrhodamine, eosins and erythrosins, cyanine dyes, e.g.Cy3 and Cy5, macrocyclic chelates of lanthanide ions, e.g. quantum dye,etc., chemilumescent dyes, e.g., luciferases, including those describedin U.S. Pat. Nos. 5,843,746; 5,700,673; 5,674,713; 5,618,722; 5,418,155;5,330,906; 5,229,285; 5,221,623; 5,182,202; the disclosures of which areherein incorporated by reference. Specific examples of where FRET assaysemploying the subject fluorescent proteins may be used include, but arenot limited to: the detection of protein-protein interactions, e.g.,mammalian two-hybrid system, transcription factor dimerization, membraneprotein multimerization, multiprotein complex formation, etc., as abiosensor for a number of different events, where a peptide or proteincovalently links a FRET fluorescent combination including the subjectfluorescent proteins and the linking peptide or protein is, e.g., aprotease specific substrate, e.g., for caspase mediated cleavage, alinker that undergoes conformational change upon receiving a signalwhich increases or decreases FRET, e.g., PKA regulatory domain(cAMP-sensor), phosphorylation, e.g., where there is a phosphorylationsite in the linker or the linker has binding specificity tophosphorylated/dephosphorylated domain of another protein, or the linkerhas Ca²⁺ binding domain. Representative fluorescence resonance energytransfer or FRET applications in which the subject proteins find useinclude, but are not limited to, those described in: U.S. Pat. Nos.6,008,373; 5,998,146; 5,981,200; 5,945,526; 5,945,283; 5,911,952;5,869,255; 5,866,336; 5,863,727; 5,728,528; 5,707,804; 5,688,648;5,439,797; the disclosures of which are herein incorporated byreference.

Another application in which the subject fluorescent proteins find useis BRET (Bioluminescence Resonance Energy Transfer). BRET is aprotein-protein interaction assay based on energy transfer from abioluminescent donor to a fluorescent acceptor protein. The BRET signalis measured by the amount of light emitted by the acceptor to the amountof light emitted by the donor. The ratio of these two values increasesas the two proteins are brought into proximity. The BRET assay has beenamply described in the literature. See, e.g., U.S. Pat. Nos. 6,020,192;5,968,750; and 5,874,304; and Xu et al. (1999) Proc. Natl. Acad. Sci.USA 96:151-156. BRET assays may be performed by genetically fusing abioluminescent donor protein and a fluorescent acceptor proteinindependently to two different biological partners to make partnerA-bioluminescent donor and partner B-fluorescent acceptor fusions.Changes in the interaction between the partner portions of the fusionproteins, modulated, e.g., by ligands or test compounds, can bemonitored by a change in the ratio of light emitted by thebioluminescent and fluorescent portions of the fusion proteins. In thisapplication, the subject proteins serve as donor and/or acceptorproteins. BRET assays can be used in many of the assays as FRET, whichassays are noted above.

The subject fluorescent proteins also find use as biosensors inprokaryotic and eukaryotic cells, e.g. as Ca²⁺ ion indicator; as pHindicator, as phorphorylation indicator, as an indicator of other ions,e.g., magnesium, sodium, potassium, chloride and halides. For example,for detection of Ca ion, proteins containing an EF-hand motif are knownto translocate from the cytosol to membranes upon Ca²⁺ binding. Theseproteins contain a myristoyl group that is buried within the molecule byhydrophobic interactions with other regions of the protein. Binding ofCa²⁺ induces a conformational change exposing the myristoyl group whichthen is available for the insertion into the lipid bilayer (called a“Ca²⁺-myristoyl switch”). Fusion of such a EF-hand containing protein toFluorescent Proteins (FP) could make it an indicator of intracellularCa²⁺ by monitoring the translocation from the cytosol to the plasmamembrane by confocal microscopy. EF-hand proteins suitable for use inthis system include, but are not limited to: recoverin (1-3),calcineurin B, troponin C, visinin, neurocalcin, calmodulin,parvalbumin, and the like. For pH, a system based on hisactophilins maybe employed. Hisactophilins are myristoylated histidine-rich proteinsknown to exist in Dictyostelium. Their binding to actin and acidiclipids is sharply pH-dependent within the range of cytoplasmic pHvariations. In living cells membrane binding seems to override theinteraction of hisactophilins with actin filaments. At pH 6.5 theylocate to the plasma membrane and nucleus. In contrast, at pH 7.5 theyevenly distribute throughout the cytoplasmic space. This change ofdistribution is reversible and is attributed to histidine clustersexposed in loops on the surface of the molecule. The reversion ofintracellular distribution in the range of cytoplasmic pH variations isin accord with a pK of 6.5 of histidine residues. The cellulardistribution is independent of myristoylation of the protein. By fusingFPs (Fluoresent Proteins) to hisactophilin the intracellulardistribution of the fusion protein can be followed by laser scanning,confocal microscopy or standard fluorescence microscopy. Quantitativefluorescence analysis can be done by performing line scans through cells(laser scanning confocal microscopy) or other electronic data analysis(e.g., using metamorph software (Universal Imaging Corp) and averagingof data collected in a population of cells. Substantial pH-dependentredistribution of hisactophilin-FP from the cytosol to the plasmamembrane occurs within 1-2 min and reaches a steady state level after5-10 min. The reverse reaction takes place on a similar time scale. Assuch, hisactophilin-fluorescent protein fusion protein that acts in ananalogous fashion can be used to monitor cytosolic pH changes in realtime in live mammalian cells. Such methods have use in high throughputapplications, e.g., in the measurement of pH changes as consequence ofgrowth factor receptor activation (e.g. epithelial or platelet-derivedgrowth factor) chemotactic stimulation/cell locomotion, in the detectionof intracellular pH changes as second messenger, in the monitoring ofintracellular pH in pH manipulating experiments, and the like. Fordetection of PKC activity, the reporter system exploits the fact that amolecule called MARCKS (myristoylated alanine-rich C kinase substrate)is a PKC substrate. It is anchored to the plasma membrane viamyristoylation and a stretch of positively charged amino acids(ED-domain) that bind to the negatively charged plasma membrane viaelectrostatic interactions. Upon PKC activation the ED-domain becomesphosphorylated by PKC, thereby becoming negatively charged, and as aconsequence of electrostatic repulsion MARCKS translocates from theplasma membrane to the cytoplasm (called the “myristoyl-electrostaticswitch”). Fusion of the N-terminus of MARCKS ranging from themyristoylation motif to the ED-domain of MARCKS to fluorescent proteinsof the present invention makes the above a detector system for PKCactivity. When phosphorylated by PKC, the fusion protein translocatesfrom the plasma membrane to the cytosol. This translocation is followedby standard fluorescence microscopy or confocal microscopy e.g. usingthe Cellomics technology or other High Content Screening systems (e.g.Universal Imaging Corp./Becton Dickinson). The above reporter system hasapplication in High Content Screening, e.g., screening for PKCinhibitors, and as an indicator for PKC activity in many screeningscenarios for potential reagents interfering with this signaltransduction pathway. Methods of using fluorescent proteins asbiosensors also include those described in U.S. Pat. Nos. 972,638;5,824,485 and 5,650,135 (as well as the references cited therein) thedisclosures of which are herein incorporated by reference.

The subject fluorescent proteins also find use in applications involvingthe automated screening of arrays of cells expressing fluorescentreporting groups by using microscopic imaging and electronic analysis.Screening can be used for drug discovery and in the field of functionalgenomics: e.g., where the subject proteins are used as markers of wholecells to detect changes in multicellular reorganization and migration,e.g., formation of multicellular tubules (blood vessel formation) byendothelial cells, migration of cells through Fluoroblok Insert System(Becton Dickinson Co.), wound healing, neurite outgrowth, etc.; wherethe proteins are used as markers fused to peptides (e.g., targetingsequences) and proteins that allow the detection of change ofintracellular location as indicator for cellular activity, for example:signal transduction, such as kinase and transcription factortranslocation upon stimuli, such as protein kinase C, protein kinase A,transcription factor NFkB, and NFAT; cell cycle proteins, such as cyclinA, cyclin B1 and cyclinE; protease cleavage with subsequent movement ofcleaved substrate, phospholipids, with markers for intracellularstructures such as endoplasmic reticulum, Golgi apparatus, mitochondria,peroxisomes, nucleus, nucleoli, plasma membrane, histones, endosomes,lysosomes, microtubules, actin) as tools for High Content Screening:co-localization of other fluorescent fusion proteins with theselocalization markers as indicators of movements of intracellularfluorescent fusion proteins/peptides or as marker alone; and the like.Examples of applications involving the automated screening of arrays ofcells in which the subject fluorescent proteins find use include: U.S.Pat. No. 5,989,835; as well as WO/0017624; WO 00/26408; WO 00/17643; andWO 00/03246; the disclosures of which are herein incorporated byreference.

The subject fluorescent proteins also find use in high through-putscreening assays. The subject fluorescent proteins are stable proteinswith half-lives of more than 24 h. Also provided are destabilizedversions of the subject fluorescent proteins with shorter half-livesthat can be used as transcription reporters for drug discovery. Forexample, a protein according to the subject invention can be fused witha putative proteolytic signal sequence derived from a protein withshorter half-life, e.g., PEST sequence from the mouse ornithinedecarboxylase gene, mouse cyclin B1 destruction box and ubiquitin, etc.For a description of destabilized proteins and vectors that can beemployed to produce the same, see e.g., U.S. Pat. No. 6,130,313; thedisclosure of which is herein incorporated by reference. Promoters insignal transduction pathways can be detected using destabilized versionsof the subject fluorescent proteins for drug screening, e.g., AP1, NFAT,NFkB, Smad, STAT, p53, E2F, Rb, myc, CRE, ER, GR and TRE, and the like.

The subject proteins can be used as second messenger detectors, e.g., byfusing the subject proteins to specific domains: e.g., PKCgamma Cabinding domain, PKCgamma DAG binding domain, SH2 domain and SH3 domain,etc.

Secreted forms of the subject proteins can be prepared, e.g. by fusingsecreted leading sequences to the subject proteins to construct secretedforms of the subject proteins, which in turn can be used in a variety ofdifferent applications.

The subject proteins also find use in fluorescence activated cellsorting applications. In such applications, the subject fluorescentprotein is used as a label to mark a population of cells and theresulting labeled population of cells is then sorted with a fluorescentactivated cell sorting device, as is known in the art. FACS methods aredescribed in U.S. Pat. Nos. 5,968,738 and 5,804,387; the disclosures ofwhich are herein incorporated by reference.

The subject proteins also find use as in vivo marker in animals (e.g.,transgenic animals). For example, expression of the subject protein canbe driven by tissue specific promoters, where such methods find use inresearch for gene therapy, e.g., testing efficiency of transgenicexpression, among other applications. A representative application offluorescent proteins in transgenic animals that illustrates this classof applications of the subject proteins is found in WO 00/02997, thedisclosure of which is herein incorporated by reference.

Additional applications of the subject proteins include: as markersfollowing injection into cells or animals and in calibration forquantitative measurements (fluorescence and protein); as markers orreporters in oxygen biosensor devices for monitoring cell viability; asmarkers or labels for animals, pets, toys, food, etc.; and the like.

The subject fluorescent proteins also find use in protease cleavageassays. For example, cleavage inactivated fluorescence assays can bedeveloped using the subject proteins, where the subject proteins areengineered to include a protease specific cleavage sequence withoutdestroying the fluorescent character of the protein. Upon cleavage ofthe fluorescent protein by an activated protease fluorescence wouldsharply decrease due to the destruction of a functional chromophor.Alternatively, cleavage activated fluorescence can be developed usingthe subject proteins, where the subject proteins are engineered tocontain an additional spacer sequence in close proximity/or inside thechromophor. This variant would be significantly decreased in itsfluorescent activity, because parts of the functional chromophor wouldbe divided by the spacer. The spacer would be framed by two identicalprotease specific cleavage sites. Upon cleavage via the activatedprotease the spacer would be cut out and the two residual “subunits” ofthe fluorescent protein would be able to reassemble to generate afunctional fluorescent protein. Both of the above types of applicationcould be developed in assays for a variety of different types ofproteases, e.g., caspases, etc.

The subject proteins can also be used is assays to determine thephospholipid composition in biological membranes. For example, fusionproteins of the subject proteins (or any other kind of covalent ornon-covalent modification of the subject proteins) that allows bindingto specific phospholipids to localize/visualize patterns of phospholipiddistribution in biological membranes also allowing colocalization ofmembrane proteins in specific phospholipid rafts can be accomplishedwith the subject proteins. For example, the PH domain of GRP1 has a highaffinity to phosphatidyl-inositol tri-phosphate (PIP3) but not to PIP2.As such, a fusion protein between the PH domain of GRP1 and the subjectproteins can be constructed to specifically label PIP3 rich areas inbiological membranes.

Yet another application of the subject proteins is as a fluorescenttimer, in which the switch of one fluorescent color to another (e.g.green to red) concomitant with the ageing of the fluorescent protein isused to determine the activation/deactivation of gene expression, e.g.,developmental gene expression, cell cycle dependent gene expression,circadian rhythm specific gene expression, and the like.

The subject non-aggregating fluorescent proteins of the subjectinvention may also be used in cell labeling applications, as describedin U.S. Application Ser. No. 60/261,448; the disclosure of which isherein incorporated by reference. For example, a fusion proteincomprising a non-aggregating protein of the invention and a member of aspecific binding partner that binds to a cell-surface molecule (e.g., aligand that binds to to a cell surface receptor; an antibody the bindsto a cell surface protein; a counterreceptor that binds to a cellsurface protein; and the like) can be used to identify and/orfractionate and/or isolate one or more cell populations from a mixtureof cells. In some embodiments, the fusion proteins multimerize, and, insome of these embodiments, cell labeling applications take advantage ofthe multimerization feature. Thus, in some embodiments, the inventionprovides intrinsically fluorescent multimeric, non-aggregating fusionprotein complex comprising a polypeptide of the present invention, andmethods for use of the fusion protein complex in cell labeling methods.

In one non-limiting example, a fusion protein comprising anantigen-binding portion of an MHC molecule, and a polypeptide of theinvention can be generated, using standard molecular biology techniques.Such fusion proteins would be expected to multimerize, but notaggregate. The multimerization property of the non-aggregating componentof the fusion protein would serve to bring together the MHC portion ofthe fusion protein, forming multimeric fusion protein complexes, and themultimeric fusion protein complex could be charged with a peptideantigen. Such fusion proteins charged with a peptide antigen could beused to identify T lymphocytes that bear on their surface a T cellreceptor specific for the same peptide antigen. Because the peptideantigens and MHC peptide-presenting domains of the fusion proteincomplexes can both be varied, the intrinsically fluorescent multimericcomplexes can be used, in general, fluorescently to label a nearlyinfinite variety of T lymphocytes based upon the antigen (and MHC)specificity of their antigen receptors.

It is, therefore, another aspect of the present invention to providemethods for using a protein of the present invention detectably to label(stain) T lymphocytes based upon the specificity of their antigenreceptors.

In a first embodiment, the method comprises contacting a T lymphocyte tobe labeled with an intrinsically fluorescent multimeric, non-aggregatingfusion protein complex comprising a polypeptide of the presentinvention, the complex having peptide antigen and MHC peptide-presentingdomains for which the antigen receptor of the T lymphocyte is specific,for a time and under conditions sufficient to permit detectable bindingof the complex to the T lymphocyte.

In the present context, a T lymphocyte is said to be specific for apeptide antigen when the affinity of its antigen receptor (TCR) for thepeptide antigen in the MHC context of the complex is sufficiently highas to confer upon the complex as a whole avidity for the T lymphocytesufficient to achieve detectable binding of the complex to TCRs on thelymphocyte surface.

By this definition, it is not impossible for a single T lymphocyte,having a single or TCR species on its surface, to be said to be specificfor a plurality of (typically closely related) peptide antigens. In suchcases, the TCR will typically have greater affinity for one of thepeptides than for the others. Often, the peptide antigen(s) for whichthe T lymphocyte is said to be specific by this definition will also becapable of stimulating cytokine expression by the T lymphocyte; such afunctional response is not, however, required, since the utility of themultimeric complexes of the present invention, as for MHC tetramers, isnot limited to labeling and identification of functionally responsive Tlymphocytes.

Conditions and times adequate for such labeling can conveniently beadapted from those used in the art for staining T lymphocytes with MHCtetramers or MHC/Ig fusions.

For example, Altman et al., Science 274:94-96 (1996) stain 200,000cytotoxic lymphocytes with MHC tetramers by incubation at 4° C. for onehour at a concentration of tetramer of approximately 0.5 mg/ml; theNIAID Tetramer facility(http://www.niaid.nih.gov/reposit/tetramer/genguide.htm) presentlyrecommends staining at each of 4° C., room temperature, and 37°, for15-60 minutes, to optimize signal to noise ratio, with decreasingincubation times used for higher temperatures. Greten et al., Proc.Natl. Acad. Sci. USA 95:7568-7573 (1998) stain 1×10⁶ peripheral bloodmononuclear cells at 4° with 3 μg of MHC class I MHC/Ig chimera.

Thus, T lymphocytes can conveniently be labeled with the intrinsicallyfluorescent multimeric complexes in the methods of the present inventionusing at least about 0.1 μg, typically at least about 0.25 μg, moretypically at least about 1 μg, 2 μg, 3 μg, 4 μg, or even at least about5 μg of multimer to label about 10⁴, 10⁵, 10⁶ or even 10⁷ peripheralblood mononuclear cells using an incubation of 15-60 minutes at atemperature between 4° C. and 37° C.

Starting with these broad, exemplary, guidelines, those skilled inlabeling T lymphocytes using MHC tetramers, MHC/Ig fusions, andfluorophore-conjugated antibodies will readily be able to determineoptimal labeling conditions. Variables that affect the amount ofmultimeric reagent to be used and the temperature and duration ofstaining include those related to the T lymphocytes—the number of Tlymphocytes in the sample that are specific for the peptide antigen (andMHC) of the complex, the total number of cells in the sample, the formof the cellular sample (e.g., whole blood, whole blood after red bloodcell lysis, Ficoll-purified peripheral blood mononuclear cell (PBMC)fraction)—and those related to the multimeric complex, including thestoichiometry and molecular weight of the labeling complex, the identityof the peptide antigen, and the choice of MHC alleles included in thecomplex.

To optimize labeling conditions, labeling reactions can readily beperformed using parallel aliquots of the cellular sample to be labeledusing a single species of multimeric complex and varying labelingconditions (e.g., temperature, duration, complex concentration, cellnumber, cell concentration, cellular purity). Negative controls caninclude labeling reactions using no multimer, using multimer lackingpeptide, and/or using multimer containing MHC peptide-presenting domainsand/or peptide antigen that will not be recognized by T lymphocytes inthe cellular sample. Effectiveness of labeling can be readily determinedfor each aliquot by flow cytometric enumeration of T lymphocytes in thesample that bind the fluorescent complex. As is well known in the flowcytometric arts, the labeled cells can be washed prior to flow cytometryto remove unbound complex from the cells and medium. All of thesetechniques and approaches are routine, and routinely performed bytechnicians, in the flow cytometric arts.

Typically, the T lymphocytes to be labeled are present within aheterogeneous sample of cells, and the goal of labeling is to detect,and often to enumerate, the antigen specific T lymphocytes within thispopulation.

Thus, in another aspect, the present invention provides methods fordetecting, in a sample of cells, T lymphocytes that are specific for achosen antigen. The method comprises contacting the sample with anintrinsically fluorescent multimeric complex according to the presentinvention, wherein the peptide antigen of the complex is the chosenantigen and the MHC presenting domains of the complex are those forwhich the T lymphocytes desired to be detected will be restricted, for atime and under conditions sufficient to permit detectable binding of thecomplex to T lymphocytes in the sample that are specific for the chosenantigen and MHC; and then detecting specific T lymphocytes in the sampleby the fluorescence of the complex bound thereto.

The detection of cell-bound fluorescence is typically performed using aflow cytometer, such as a FACSVantage™, FACSVantage™ SE, or FACSCaliburflow cytometer (Becton Dickinson Immunocytometry Systems, San Jose,Calif., USA). The lasers chosen for excitation will be determined by theabsorption spectrum of the multimeric complex and of any additionalfluorophores desired to be detected concurrently in the sample. Forexample, if the multimeric complex has the spectral characteristics ofnative DsRed—e.g., a homotetramer in which the fusion protein subunitsall have the native DsRed GFP-like chromophore—a standard argon ionlaser with 488 nm line can be used for excitation. For detection, thefilter sets and detector types will be chosen according to the emissionspectrum of the multimeric complex and of any additional fluorophoresdesired to be detected in the sample. For example, if the multimericcomplex has the spectral characteristics of native DsRed, with emissionmaximum at about 583 nm, fluorescence emission from the complex can bedetected in the FL2 channel using a PE setup.

Alternatively, cell-bound complex fluorescence can be detected using amicrovolume fluorimeter, such as the IMAGN 2000 (Becton DickinsonImmunocytometry Systems, San Jose, Calif., USA). Applications ofmicrovolume fluorimetry to, and conditions for, characterization ofblood cell are described, inter alfa, in Seghatchian et al., Transfus.Sci. 22(1-2):77-9 (2000); Glencross et al., Clin. Lab. Haematol.21(6):391-5 (1999); and Read et al., J. Hematother. 6(4):291-301 (1997).

Alternatively, cell-bound complex fluorescence can be detected using alaser scanning cytometer (Compucyte Corp., Cambridge, Mass., USA).

Cell-bound fluorescence of the multimeric complex can also be detecteddirectly on a microscope slide, whether from touch prep, cytospin prep,or tissue sample, using conditions essentially as described in Skinneret al., “Cutting edge: In situ tetramer staining of antigen-specific Tcells in tissues,” J. Immunol. 165(2):613-7 (2000).

The T lymphocyte-containing sample can be a whole blood sample,typically a peripheral venous blood specimen drawn directly into ananticoagulant collection tube (e.g., EDTA-containing orheparin-containing Vacutainer™ tube, Becton Dickinson VacutainerSystems, Franklin Lakes, N.J., USA).

Advantageously, the T lymphocyte-containing sample can also be a wholeblood sample that has been treated before detection with a red bloodcell (RBC) lysing agent as is described, inter alia, in Chang et al.,U.S. Pat. Nos. 4,902,613 and 4,654,312; lysing agents are well known inthe art and are available commercially from a number of vendors (FACS™Lysing Solution, Becton Dickinson Immunocytometry Systems, San Jose,Calif., USA; Cal-Lyse™ Lysing Solution, Caltag Labs, Burlingame, Calif.,USA; No-Wash Lysing Solution, Beckman Coulter, Inc., Fullerton, Calif.).The sample can optionally be washed after RBC lysis and beforedetection.

The sample within which T lymphocytes are desired to be detectedaccording to the methods of the present invention can also be aperipheral blood fraction, advantageously a mononuclear cell (PBMC)fraction. PBMCs can be prepared according to any of the well-knownart-accepted techniques, including centrifugation through Ficoll-Paque(Amersham Pharmacia Biotech, Piscataway, N.J., USA) and centrifugationdirectly in a cell preparation blood collection tube (Vacutainer™ CPT™Cell Preparation Tube, Becton Dickinson, Franklin Lakes, N.J., USA).

The sample can also advantageously be a sample enriched in Tlymphocytes, e.g. cultured lymphocytes (as from a clonal cell line ormulticlonal culture), lymphocytes extracted or eluted from a tissue withlymphocytic infiltrate (e.g., tumor infiltrating lymphocytes extractedfrom a tumor biopsy and optionally expanded in culture), lymphocytesdrawn from lymphatics or thymus, or lymphocytes obtained after a firstround of fluorescence-activated cell sorting.

In another embodiment, the method further comprises enumerating theantigen-specific T lymphocytes so detected. Enumeration can convenientlybe expressed in the form of a total cell count, percentage ofantigen-specific T lymphocytes among cells assayed (either total,mononuclear, or T lymphocytic), or percentage of antigen-specificlymphocytes in a T cell subset.

For several of these metrics, it is necessary additionally to quantitatethe total number of T lymphocytes within the sample as a whole, or thetotal number of T lymphocytes of a particular subset within the sampleas a whole.

Thus, in other embodiments, the method of the present invention furthercomprises contacting the sample with at least one fluorophore-conjugatedantibody, the antibody selected from the group consisting of pan-Tantibodies and T cell subsetting antibodies, and then detectingfluorescence concurrently from the multimeric fluorescent complex andfrom the fluorophore-conjugated antibodies.

Antibodies usefully used in this embodiment include antibodies specificfor CD3, CD4, CD8, CD45RO, CD45RA, and CD27. As would be understood, theantibodies would typically be specific for the marker as expressed bythe taxonomic species (human, mouse, rat, etc.) whose T lymphocytes arebeing detected, or would be cross-reactive therewith.

As is well known in the flow cytometric arts, the pan-T and/or Tlymphocyte subsetting antibodies can be used for any or all oftriggering data acquisition, live gating, or gating prior-acquired data.

It is often advantageous in the methods of the present invention toacquire a large number of events since, in many samples,antigen-specific T lymphocytes occur infrequently. In addition, it ispossible at times to improve the signal to noise ratio for detectingantigen-specific T lymphocytes by triggering or gating on fluorescencefrom antibodies specific for T lymphocyte activation antigens.

Thus, in another embodiment, the method further comprises contacting thesample with at least one fluorophore-conjugated antibody specific for aT cell activation antigen, and then detecting fluorescence concurrentlyfrom the multimeric fluorescent complex and from thefluorophore-conjugated antibodies. The antibodies can usefully bespecific for an activation antigen selected from the group consisting ofCD69, CD25, CD71 and MHC class II (for labeling human T lymphocytes,HLA-DR).

Advantageously, the antibodies used in the methods of the presentinvention will be prior-conjugated directly to a fluorophore, typicallya fluorophore whose emission is flow cytometrically distinguishable fromthat of the intrinsically fluorescent multimeric T cell labeling complexand from that of other fluorophores concurrently used in the method.Fluorophores can usefully be selected from the group consisting offluorescein isothiocyanate (FITC), phycoerythrin (PE), peridininchlorophyll protein (PerCP), allophycocyanin (APC), Texas Red, AlexaFluor 488 (Molecular Probes, Inc., Eugene Or.), and the tandemfluorophores PerCP-Cy5.5, PE-Cy5, PE-Cy7, and APC-Cy7. Antibodies canalso usefully be conjugated to biotin, permitting second stage detectionusing fluorophore-labeled streptavidin.

The methods of the present invention for detecting and enumeratingantigen-specific T lymphocytes can be used for the same purposes as areprior art methods, including use of MHC tetramers and MHC/Ig chimeras,as well as other functional assays, such as limiting dilution assay,ELISPOT, and flow cytometric detection of intracellular cytokineexpression (Waldrop et al., J. Clin. Invest. 99(7):1739-50 (1997)). Themethods can thus be used, e.g., to assess CD4⁺ and CD8⁺ T cell responsesto infection, to vaccines, and in autoimmunity.

Depending upon the instrument used, detection of antigen-specific Tlymphocytes can be coupled directly or indirectly to their sorting, thusproviding, in other aspects of the invention, methods for enriching andfor depleting a sample for T lymphocytes that are specific for a chosenantigen.

In general, the method comprises contacting the sample with anintrinsically fluorescent multimeric complex of the present invention,wherein the peptide antigen of the complex is the chosen antigen and theMHC presenting domains of the complex are those for which the Tlymphocytes desired to be enriched or depleted will be restricted, for atime and under conditions sufficient to permit detectable binding of thecomplex to T lymphocytes in the sample that are specific for the chosenantigen and MHC. After binding, labeled T lymphocytes are enriched ordepleted based upon the fluorescence of the complex bound thereto.

Such methods are conveniently performed using a fluorescence activatedcell sorter: sorting based at least in part upon fluorescence from themultimeric complex of the present invention directly depletes the samplefrom which the cells are removed and enriches the aliquot into which thecells are placed.

It is possible, however, to use the multimers of the present inventionto enrich or deplete cells using approaches other thanfluorescence-activated cell sorting.

For example, T lymphocytes stained specifically with the multimer can beseparated magnetically, rather than fluorimetrically, by furtherconjugation of the TCR-bound complex to a superparamagnetic particle.This can be done, e.g., using an antibody specific for an epitope of thefusion protein (e.g., where DsRed contributes GFP-like chromophoreand/or multimerization domains, the antibody can be the DsRed-specificantibody available commercially from Clontech Labs, Palo Alto, Calif.,USA).

As another example, T lymphocytes stained specifically with the multimercan be separated using biotin/avidin affinity interactions, rather thanfluorescence, by further conjugation of the TCR-bound complex to biotin,followed by use of an avidin affinity matrix. This further labeling ofthe multimeric complex can be done indirectly using a biotin-conjugatedantibody specific for an epitope of the fusion protein. Alternatively,the multimer can itself be prior-conjugated to biotin, either chemicallyor, upon engineering of a BirA substrate peptide into the complex(typically the fusion protein), enzymatically.

Samples enriched in antigen-specific T cells according to the methods ofthe present invention can be used in vitro for study of specificinteractions of antigen-specific T cells with antigen-presenting cells,cytotoxic targets, B cells, or other cellular elements of the immunesystem. Samples enriched in antigen-specific T cells according to themethod of the present invention can also be used in vitro to modify suchinteractions. See, e.g., Dal Porto et al., et al., Proc. Natl. Acad.Sci. USA 90:6671-6675 (1993).

Samples enriched in antigen-specific T cells according to the methods ofthe present invention can also be used for in vivo therapeuticintervention, such as for tumor immunotherapy. See, e.g., Oelke et al.,Clin. Cancer Res. 6(5):1997-2005 (2000).

The antibodies of the subject invention, described above, also find usein a number of applications, including the differentiation of thesubject proteins from other fluorescent proteins.

Kits

Also provided by the subject invention are kits for use in practicingone or more of the above described applications, where the subject kitstypically include elements for making the subject proteins, e.g., aconstruct comprising a vector that includes a coding region for thesubject protein. The subject kit components are typically present in asuitable storage medium, e.g., buffered solution, typically in asuitable container. Also present in the subject kits may be antibodiesto the provided protein. In certain embodiments, the kit comprises aplurality of different vectors each encoding the subject protein, wherethe vectors are designed for expression in different environments and/orunder different conditions, e.g., constitutive expression where thevector includes a strong promoter for expression in mammalian cells, apromoterless vector with a multiple cloning site for custom insertion ofa promoter and tailored expression, etc.

In addition to the above components, the subject kits will furtherinclude instructions for practicing the subject methods. Theseinstructions may be present in the subject kits in a variety of forms,one or more of which may be present in the kit. One form in which theseinstructions may be present is as printed information on a suitablemedium or substrate, e.g., a piece or pieces of paper on which theinformation is printed, in the packaging of the kit, in a packageinsert, etc. Yet another means would be a computer readable medium,e.g., diskette, CD, etc., on which the information has been recorded.Yet another means that may be present is a website address which may beused via the internee to access the information at a removed site. Anyconvenient means may be present in the kits.

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTAL I. Sequences of Parental Aggregating Wild-Type AnthozoanProteins and Mutants Thereof

The following table summarizes the properties of nine specific wild typeAnthozoan proteins of the subject invention:

TABLE I Amino Acid Nucleotide Sequence Sequence NFP Species IdentifierFigure ID No. ID No. 1 Anemonia majano amFP486 1 02 01 3 Zoanthus sp.zFP506 2 04 03 4 Zoanthus sp. zFP538 3 06 05 6 Discosoma drFP583 4 08 07sp. “red” 7 Anemonia sulcata asFP600 5 10 09 8 6/9Q drFP583/d 6 12 11mFP592

II. Mutagenesis

Site-directed mutagenesis was performed by PCR with primers containingappropriate target substitutions. All mutants were cloned between BamHIand HindIII restriction sites of the pQE30 vector (Qiagen). Recombinantproteins were 6× Histidine-tagged to contain the sequence ‘MRHHHHHHGS’instead of the first Met. After overnight expression in E. coli,fluorescent proteins were purified using TALON Metal Affinity Resin(CLONTECH). SDS-PAGE analyses revealed that proteins were at least 95%pure.

In greater detail, mutagenesis was performed by the overlap extensionmethod (Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerasechain reaction. (Gene 1989, 77, 51-59). Briefly, two overlappingfragments of FP coding region were amplified. “Forward cloning” and“reverse mutagenesis” primers were used for 5′-end fragmentamplification, and “forward mutagenesis” and “reverse cloning” primerswere used for 3′-end fragment amplification. PCR was carried out usingAdvantage® 2 Polymerase Mix (CLONTECH) in 1× manufacturer's buffersupplemented with 100 μM of each dNTP, 0.2 μM of each primer and 1 ng ofplasmid DNA in 25 μl (final volume). The cycling parameters were set at:95° C. for 10 seconds, 65° C. for 30 s, 72° C. for 30 s. 20 cycles werecompleted using PTC-200 MJ Research thermocycler. To remove plasmidsencoding initial (wild type) protein, the 5′- and 3′-fragments wereexcised from 2% low-melting agarose gel in 1×TAE buffer. To drain theDNA solution, the gel pieces were subjected to 3 freeze-thaw cycles. 5′-and 3′-fragments were combined to obtain full-length cDNA as follows.Equal volumes of 5′-fragment solution, 3′-fragment solution and 3×PCRmixture containing Advantage 2 Polymerase Mix, buffer and dNTPs weremixed together and subjected to 2-3 cycles of 95° C. for 20 s, 65° C.for 30 min, 72° C. for 30 s. Then, the reaction was diluted 10 fold and1 μl of the diluted sample was used as a template for PCR with forwardand reverse cloning primers (as described above for 5′- and 3′-fragmentsamplification). As a result, ready-for-cloning fragments containingfull-length coding regions with target substitution(s) were generated.

Mutant cDNAs were digested with BamHI and HindIII (the cloning primerscontain sites for these endonucleases), and then cloned into pQE30(Qiagen) digested with BamHI and HindIII. Recombinant proteins contained6×His tag on the N-terminus.

Selected E. coli clones were grown at 37° C. in 50 ml to an opticaldensity of (OD) 0.6. At that point, the expression of recombinant FP wasinduced with 0.2 mM IPTG. The cultures were then incubated overnight.The following day, cells were harvested by centrifugation, resuspendedin buffer (20 mM Tris-HCl, pH 8.0; 100 mM NaCl), and disrupted bysonication. Fluorescent proteins were purified from the soluble fractionusing TALON Metal Affinity Resin (CLONTECH). Proteins were at least 95%pure according to SDS-PAGE.

III. Mutants Generated Table 2 Provides Details Regarding RepresentativeNon-Aggregating Mutants Generated Using the Above Protocol

Non- Differences Target Aggregating between wild-type substitutions inMutant Wild type Parental protein and non-aggregating Figure Designatorprotein mutant parental mutant mutant Seq ID NOs NFP6-NA drFP583 “E57”(V105A, Faster and more R2A, K5E, K9T FIG. 7 E57-NA I161T, S197A)complete folding DsRED2 in E. coli SEQ ID NO. 13 E5-NA drFP583 “Timer”Change color with R2A, K5E, K9T FIG. 8 Timer-NA (V105A, time S197T) SeqID NO: 14 6/9Q-NA dsFP593/ ds/drFP616 Red-shifted - S2del, C3del,drFP583 fluorescence K5E, K9T NFP3-NA zFP506 N66M 1.8-fold increase K5E,K10E FIG. 9 in brightness in E. coli SEQ ID NOs: 15 & 16 NFP4-NA zFP538M129V More complete K5E, K9T FIG. 10 folding in E. coli SEQ ID NOs: 17 &18 NFP1-NA amFP486 K68M 1.5-fold increase K6E FIG. 11 in brightness inE. coli SEQ ID NOs: 19 & 20 NFP7-NA asFP595 “M35-5” (F4L, 5-foldincrease in K6T, K7E FIG. 12 M35-5NA K12R, F35L, brightness in E. T68A,F84L, coli, in SEQ ID NOS: 21 A143S, comparison with & 22 K163E, mutantT68A, M202L) A143S [3] NFP7-NA asFP595 “M35-5” (F4L, K6T, K7E FIG. 13dimer K12R, F35L, M35-5 dimer- T68A, F84L, SEQ ID NOS: 23 NA A143S, & 24K163E, M202L)FIG. 14 provides an alignment of certain non-aggregating mutants asdescribed above.

IV. Characterization of Representative Non-Aggregating Mutants A.Materials and Methods Theoretical pI

Theoretical pI were calculated using ProtParam program available on Website which is produced by putinng: “http://” before and “.html” after“expasy.pku.edu.cn/tools/protoparam” and described in Appel R. D.,Bairoch A., Hochstrasser D. F. A new generation of information retrievaltools for biologists: the example of the ExPASy WWW server. TrendsBiochem. Sci. 1994, 19:258-260.

Methods of Evaluating Protein Aggregation

1. “Pseudo-native” protein electrophoresis. For fast evaluation ofaggregation properties of the mutant proteins we used a simple methodwhich we term “pseudo-native” protein electrophoresis. This method isbased on applying on a common sodium dodecyl sulfate-polyacrylamide gel(SOS-FAG) the non-boiled protein samples. In these conditions, FPsremain fluorescent. In addition, these conditions maintain thesuper-molecular structure of applied proteins. High-molecular weightaggregated proteins remain at the top of the gel, while tetramerproteins migrate as a band >100 kD.2. Light scattering. This method is based on light scattering insolution by particles of aggregated protein. The larger size and amountof such particles, the greater the light scattering. As light scatteringdepends on the wavelength of light (scattering is much more pronouncedfor short waves), the aggregation results in general slope of absorptionspectrum. In general, it is preferable to evaluate aggregation by theratio of absorption at a shorter wavelength (where absorption ofnon-aggregated protein is minimal) to absorption at a longer wavelength(where absorption of non-aggregated protein is maximal). For instance,for E57 one might evaluate aggregation by measuring the ratio ofabsorption as follows: absorption(400 nm)/absorption(566 nm). In anon-aggregated protein sample, this ratio should be close to zero.

The mutants were tested under the same buffer conditions, whichconditions do not prevent aggregation, at concentrations of 1 mg/ml.

3. Brightness in mammalian cell line. Particular mutants weretransiently transfected into the mammalian cell line Phoenix using C1vector (CLONTECH). Aggregation was evaluated by visual inspection of thenumber of cells expressing FP using fluorescent microscope.4. Exact kinetics of aggregation in vivo is unclear because it isdifficult to measure this process within living cells. Probably,aggregation depends on FP concentration since brighter cells usuallydisplay more pronounced FP aggregates. Nevertheless, aggregation picturecan be observed even in low fluorescent cells as soon as the signalbecome visible. This indicates that threshold value of FP concentrationsufficient for aggregation is rather low.

Aggregation of purified FPs is additionally observed in vitro. Forexample, almost all Anthozoa FPs partially precipitate from solution(PBS) without any loss of color or fluorescence. To visualizeaggregation of purified FPs, we used “pseudo-native” proteinelectrophoresis, based on the discontinuous SDS-PAGE of non-heatedprotein samples (Baird, G. S., Zacharias, D. A. and Tsien, R. Y. (2000)Proc. Natl. Acad. Sci. U.S.A. 97, 11984-11989). Under these conditions,FPs retain not only fluorescent properties, but also super-molecularstructure: high-molecular weight-aggregated proteins remain at the topof the gel, while oligomers migrate as bands of the high molecularweights.

B. Results

Red fluorescent drFP583 was the first protein subjected to mutagenesis.An improved mutant of DsRed (the commercially available version ofdrFP583 with altered codon usage optimized for expression in mammaliancells), designated E57, was used as a parental gene (Table 2). Mutantsof E57 containing the substitutions R2A, K5E, K9T (in differentcombinations) were generated. After expression in E. coli andpurification, these proteins were analyzed by pseudo-native PAGE (seeabove). All mutants displayed lower levels of aggregation in comparisonwith parental E57, and the R2A substitution appeared to have thestrongest impact on this outcome (not shown). A mutant, denoted E57-NA,containing all three substitutions (R2A, K5E, and K9T) that showed noaggregation (FIG. 15) and was very similar to E57 in terms ofexcitation-emission maxima, fluorescence brightness and maturationspeed, was selected as the optimal protein.

E57-NA displayed an excellent fluorescence image in mammalian cells(FIG. 16). In most cells, nuclei and nucleoli were clearly visible, andcell borders and processes were well-defined. In contrast, thefluorescence of E57-expressing cells was smeared, with no visibleintracellular structures. The borders of the fluorescent signal oftendid not coincide with cell borders and processes, so that cells lookedrounded. Therefore, both in vivo and in vitro tests confirmed that theE57-NA protein was low-aggregating. Importantly, pilot trials of E57-NAin other laboratories showed greatly decreased toxicity of this proteinin comparison with both DsRed and E57, during expression in cell lines(B. Angres, Clontech, Palo-Alto, Calif., USA, personal communication),Xenopus embryos (A. Zaraisky, IBCh, Moscow, Russia, personalcommunication), and plants (A. Touraev, IMG, Vienna Biocenter, Vienna,Austria, personal communication). Now E57-NA is commercially availablefrom Clontech as DsRed2.

Recently, an interesting mutant of DsRed entitled ‘Timer’, because itchanges color with time, was described. To generate a non-aggregatingversion of this protein, we employed the three substitutions mentionedabove. The novel mutant, Timer-NA, was generated (Table 2), whichpossessed practically the same maturation properties with color changeas parental Timer, but did not form aggregates on pseudo-native PAGE(FIG. 15). In mammalian cells, the differences between Timer andTimer-NA were analogous to those between E57 and E57-NA (FIG. 16).

The next FP targeted was ds/drFP616 (6/9Q), which displays red-shiftedfluorescence with a peak at 616 nm. The protein was generated by theshuffling of two red FPs (dsFP593 and drFP583), followed by randommutagenesis. However, ds/drFP616 showed extremely high aggregation. Themutation of two Lys residues at positions 5 and 9 at the N-terminalregion of ds/drFP616 as for E57 and Timer (FIG. 14, Table 2), resultedin a significant decrease in the amount of aggregated protein onpseudo-native PAGE, although residual aggregation was still detected inthe Lys mutant, ds/drFP616-K5E/K9T (not shown). After the screening ofE. coli clones producing protein, one was selected that displayedcomplete absence of aggregation on pseudo-native PAGE (FIG. 15).Sequence analyses revealed that this clone contains two additionalmutations in the N-terminal region of the protein (Ser-2 and Cys-3,deleted accidentally during cloning procedures). When expressed ineukaryotic cells, the mutant, designated ds/drFP616-NA (6/9QNA), showedsignificant improvement in fluorescence image, similar to E57-NA andTimer-NA. In contrast to the bright but completely unstructuredblot-like image of parental ds/drFP616, ds/drFP616-NA was more evenlydistributed in nuclei and cytoplasm (FIG. 16).

The mutagenesis strategy described above was subsequently applied tofour FPs of different colors: green zFP506, yellow zFP538, red asFP595,and cyan amFP486 proteins, based on the improved mutants generatedearlier by random mutagenesis (Table 2). When expressed in E. coli,mutant proteins displayed greater brightness and faster and morecomplete protein folding, in comparison with corresponding wild typeproteins (unpublished data). However, the introduced substitutions hadno influence on the aggregation properties of these FPs. In an attemptto decrease aggregation tendency, all lysines near the N-termini of theproteins were mutated (FIG. 14, Table 2). In vitro analyses of resultingsecondary protein mutants confirmed no aggregation (FIG. 15).Additionally, all four non-aggregating mutants (zFP506-N66M-NA,zFP538-M129V-NA, amFP486-K68M-NA, and asFP595-M35-5-NA) displayed clearimprovement in fluorescence images in mammalian cells, analogous toE57-NA, Timer-NA, and ds/drFP616-NA (FIG. 16, images for amFP486 andasFP595 are not shown).

C. Conclusion

In summary, we conclude that basic residues near the N-termini ofAnthozoa FPs play a prominent role in the formation of proteinaggregates. A number of examples of the significant effect of singleamino acid substitutions on protein aggregation are documented.Similarly, substitution of one to three residues in FPs led to aconsiderable increase in protein solubility.

V. Additional Characterizations

TABLE 3 Properties of E57-based mutants with altered pl. AggregationVisual Substitutions in vitro Aggregation brightness (compared withVisual (pseudo- in vitro (light in Aggregation E57) Theoreticalbrightness native scattering eukaryotic in eukaryotic N See FIG. 4 pl inE. coli PAGE) ^(a) test) ^(b) cell culture cell culture 1 “E57” 7.78high High 1 high high 2 Q137E, Q188E 6.72 high Low 0.05 low medium 3R2del, Q137E, 6.46 high Very low 0.03 medium low Q188E 4 R2del, Q137E,6.72 high High NT low high Q188EI180A, M182K 5 R2A, K5E, K9T 6.08 highVery low 0.025 high very low 6 R2del, R13S, R17E 6.08 high Low NT lowlow 7 T21D 6.72 medium High NT NT NT 8 R36D 6.46 high Medium NT NT NT 9R2A, K5E, K9T, 5.65 high Very low 0.02 low low R13S, R17E ^(a) Thismethod is based on applying on a SDS-PAGE the non-boiled proteinsamples. In these conditions FPs are fluorescent and the conditions ofelectrophoresis preserve super-molecular structure. ^(b) Ratio:(Absorption (400 nm)-Absorption (650 nm)/(Absorption (566 nm)-Absorption(650 nm)).

It is evident from the above discussion and results that the subjectinvention provides important new mutant fluorescent proteins and nucleicacids encoding the same, where the subject mutants have improvedfeatures, i.e., non-aggregation, when compared with a referencechromo/fluoroprotein, and where the subject proteins and nucleic acidsfind use in a variety of different applications. As such, the subjectinvention represents a significant contribution to the art.

All publications and patent applications cited in this specification areherein incorporated by reference as if each individual publication orpatent application were specifically and individually indicated to beincorporated by reference. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present invention is not entitled to antedate suchpublication by virtue of prior invention.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it is readily apparent to those of ordinary skill in theart in light of the teachings of this invention that certain changes andmodifications may be made thereto without departing from the spirit orscope of the appended claims.

1-20. (canceled)
 21. A nucleic acid molecule, comprising: a nucleotidesequence encoding a functional non-aggregating fluorescent protein whoseamino acid sequence differs from the amino acid sequence of dsRed (SEQID NO:8) by at least one negatively charged or neutral amino acidsubstitution corresponding to a basic residue within the first 25N-terminal residues of dsRed.
 22. The nucleic acid molecule according toclaim 21, wherein the amino acid substitution is a substitutioncorresponding to residue 2, 5, or 9 of dsRed.
 23. The nucleic acidmolecule according to claim 22, wherein the nucleic acid moleculeencodes a protein comprising an alanine at the residue corresponding toresidue 2 of dsRed, a glutamic acid at the residue corresponding toresidue 5 of dsRed, or a threonine at the residue corresponding toresidue 9 of dsRed.
 24. The nucleic acid molecule according to claim 21,wherein the functional mutant fluorescent protein has a reducedaggregation capacity relative to the aggregation capacity of dsRed. 25.The nucleic acid molecule according to claim 21, wherein the functionalmutant fluorescent protein does not aggregate.
 26. A constructcomprising a vector and the nucleic acid according to claim
 21. 27. Anexpression cassette comprising: (a) a transcriptional initiation regionfunctional in an expression host; (b) the nucleic acid moleculeaccording to claim 21; and (c) a transcriptional termination regionfunctional in the expression host.
 28. An isolated host cell comprisingan expression cassette according to claim 27 as part of anextrachromosomal element or integrated into the genome of a host cell asa result of introduction of the expression cassette into the host cell.29. A transgenic cell or the progeny thereof comprising a transgene thatis a nucleic acid according to claim
 21. 30. A transgenic organismcomprising a transgene that is a nucleic acid according to claim
 21. 31.An antibody biding specifically to a protein according to claim
 21. 32.A method of producing a chromo- or fluorescent mutant protein, themethod comprising: growing a cell according to claim 28 to express theprotein; and isolating the protein.
 33. A kit comprising the nucleicacid according to claim
 21. 34. A nucleic acid molecule, comprising: anucleotide sequence encoding a functional non-aggregating fluorescentprotein, wherein the nucleotide sequence differs from the nucleotidesequence of SEQ ID NO:7 (dsRed) by at least one codon which encodes fora negatively charged or neutral amino acid at a residue corresponding toa basic residue within the first 25 N-terminal residues of thepolypeptide encoded by SEQ ID NO:7.
 35. The nucleic acid moleculeaccording to claim 34, wherein the codon encodes for a negativelycharged or neutral amino acid at a residue corresponding to residue 2,5, or 9 of the polypeptide encoded by SEQ ID NO:7.
 36. The nucleic acidmolecule according to claim 35, wherein the codon encodes for an alanineat corresponding residue 2, a glutamic acid at corresponding residue 5,or a threonine at corresponding residue
 9. 37. The nucleic acid moleculeaccording to claim 34, wherein the functional mutant fluorescent proteinhas a reduced aggregation capacity relative to the aggregation capacityof dsRed.
 38. The nucleic acid molecule according to claim 34, whereinthe functional mutant fluorescent protein does not aggregate.
 39. Afunctional mutant fluorescent protein comprising an amino acid sequencethat differs from the amino acid sequence of the aggregating fluorescentprotein dsRed (SEQ ID NO:8) by at least one negatively charged orneutral amino acid substitution corresponding to a basic residue withinthe first 25 N-terminal residues of dsRed.
 40. The functional mutantfluorescent protein of claim 38, wherein the negatively charged orneutral amino acid substitution is a substitution corresponding toresidue 2, 5, or 9 of dsRed.